IL-2 fusion proteins with modulated selectivity

ABSTRACT

The invention provides cytokine fusion proteins with an increased therapeutic index, and methods to increase the therapeutic index of such fusion proteins. The fusion proteins of the invention are able to bind to more than one type of cytokine receptor expressed on cells and also bind to more than one cell type. In addition, the fusion proteins of the invention exhibit a longer circulating half-life in a patient&#39;s body than the corresponding naturally occurring cytokine.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of U.S. Ser. No.60/337,113, filed Dec. 4, 2001, and U.S. Ser. No. 60/371,966, filed Apr.12, 2002, the entire disclosures of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates generally to fusion proteins containing acytokine, and methods to increase the therapeutic effectiveness of suchfusion proteins. More specifically, the present invention relates tocytokine fusion proteins that exhibit a longer circulating half-life ina patient's body than the corresponding naturally occurring cytokine andthat have improved therapeutic properties. In particular, the inventionrelates to IL2 fusion protein with improved therapeutic characteristics.

BACKGROUND

Interleukin-2 (IL-2) is a potent cytokine that acts on the immune systemto generate primarily a cell-mediated immune response. Under theappropriate conditions, IL-2 is produced locally at high concentrationsnear the site of an antigen in order to supply the necessaryco-stimulatory signals for generating an immune response to the antigen.Because of its role in the growth and differentiation of T cells, IL-2has been a candidate in immunotherapeutic approaches to treating tumors.In addition to stimulating T cells, IL-2 has also been shown tostimulate B cells, NK cells, lymphokine activated killer cells (LAK),monocytes, macrophages and dendritic cells.

IL-2 is an approved therapeutic agent for the treatment of metastaticrenal carcinoma and metastatic melanoma but its use is restricted due tosevere toxic side effects, which include fever, nausea, vascular leakageand hypotension. Among the various toxic effects observed with IL-2administration, the one toxic effect that is the least desirable and isbelieved to substantially interfere with IL-2 therapy is vascular leaksyndrome (VLS) and the complications associated with it.

Therefore, there remains a need in the art to further enhance thetherapeutic usefulness of IL-2 proteins.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the identification ofmutations in the IL-2 moiety of an IL-2 fusion protein to increase themaximum tolerated dose of the protein relative to the dose of maximaleffectiveness for that protein when administered to a patient. Preferredfusion proteins are able to bind by distinct interactions to more thanone receptor species expressed on the same cell in the patient's body.Preferred cytokine fusion proteins include a cytokine that is able tobind to more than one type of cytokine receptor complex and to more thanone cell type. The invention also provides methods to identifyparticular cytokine fusion protein variants with useful properties.

The present invention provides fusion proteins comprising a non-IL-2moiety fused to a mutant IL-2 moiety, where the fusion protein exhibitsa greater selectivity than a reference protein including the non-IL-2moiety fused to a non-mutant IL-2 moiety, and where the selectivity ismeasured as a ratio of activation of cells expressing IL-2Rαβγ receptorrelative to activation of cells expressing IL-2Rβγ receptor.

The mutant IL-2 moiety of the fusion proteins includes a mutation in oneor more amino acids of the mature human IL-2 protein. In one embodiment,fusion proteins according to the invention include an amino acidsubstitution at one or more amino acid positions in the IL-2 moiety. Inanother embodiment, fusion proteins of the invention include deletionsof amino acids at one or more amino acid positions in the IL-2 moiety.In yet another embodiment, fusion proteins of the invention includemodifications of one or more amino acids in the IL-2 moiety of thefusion proteins.

Mutations in the fusion proteins of the invention alter the selectivityof fusion proteins relative to a reference fusion protein, where theselectivity is measured as a ratio of activation of cells expressingIL-2Rαβγ receptor relative to activation of cells expressing IL-2Rβγreceptor. Mutations in the fusion proteins can also result in adifferential effect on the fusion protein's affinity for IL-2Rβγreceptor relative to the fusion protein's affinity for IL-2Rαβγreceptor. Preferred mutations or alterations reduce a fusion protein'sactivation of cells expressing IL-2Rβγ receptor relative to the fusionprotein's activation of cells expressing IL-2Rαβγ receptor.

Preferred fusion proteins of the invention generally exhibit adifferential effect that is greater than about 2-fold. In one aspect,the differential effect is measured by the proliferative response ofcells or cell lines that depend on IL-2 for growth. This response to thefusion protein is expressed as an ED50 value, which is obtained fromplotting a dose response curve and determining the protein concentrationthat results in a half-maximal response. The ratio of the ED50 valuesobtained for cells expressing IL-2Rβγ receptor to cells expressingIL-2Rαβγ receptor for a fusion protein of the invention relative to theratio of ED50 values for a reference fusion protein gives a measure ofthe differential effect for the fusion protein.

The selectivity of fusion proteins of the invention may be measuredagainst a reference fusion protein comprising the same non-IL-2 moietyas in the fusion protein fused to a non-mutant IL-2 moiety. In apreferred embodiment, a differential effect measured for the fusionproteins of the invention, as described above, is between about 5-foldand about 10-fold. Preferably, the differential effect exhibited by thefusion proteins of the invention is between about 10-fold and about1000-fold.

In an alternative preferred embodiment, the selectivity of the fusionprotein is compared to the selectivity of a reference fusion proteinthat comprises the same non-IL-2 moiety as in the fusion protein fusedto an IL-2 moiety including mature human IL-2 with an amino acidsubstitution at position 88 changing an asparagine to an arginine(N88R). Fusion proteins of the invention that have an improvedtherapeutic index include fusion proteins having a selectivity close tothat of N88R but between about 0.1% to about 100% of the selectivity ofa reference fusion protein with the N88R amino acid substitution. Inanother embodiment, fusion proteins of the invention have a selectivitybetween about 0.1% to about 30% of the selectivity of a reference fusionprotein with the N88R amino acid substitution in the IL-2 moiety. Fusionproteins of the invention also include fusion proteins that have aselectivity between about 1% to about 20% of the selectivity of thereference fusion protein with the N88R amino acid substitution in theIL-2 moiety. Selectivity of fusion proteins of the invention can also bebetween about 2% to about 10% of the selectivity of the reference fusionprotein including the N88R amino acid substitution in the mature humanIL-2 moiety.

Fusion proteins of the invention have a serum half-life that is longerthan the serum half life of mature human IL-2 protein. The long serumhalf-life of fusion proteins of the invention can be attributed to thenon-IL-2 moiety of the fusion protein. For example, in one embodiment,the non-IL-2 moiety of a fusion protein of the invention is albumin. Inanother embodiment, the non-IL2 moiety of a fusion protein of theinvention is an antibody domain including, for example, variants of theKS-1/4 antibody domain, variants of the NHS76 antibody domain andvariants of the 14.18 antibody domain. The antibody domain can also beselected from a variety of other antibodies, for example, antibodiesagainst various tumor and viral antigens.

In a preferred embodiment, a differential effect measured for the fusionproteins of the invention, as described above, is between about 5-foldand about 10-fold. Preferably, the differential effect exhibited by thefusion proteins of the invention is between about 10-fold and about1000-fold.

It is useful to mutate amino acids in the IL-2 moiety of fusion proteinsof the invention that result in a differential effect which is 2-fold orgreater. Different amino acid mutations in the IL-2 moiety result in adifferential effect greater than about 2-fold, between about 5-fold andabout 10-fold, or preferably between about 10-fold and about 1000-fold.In a preferred embodiment, the amino acid mutation is a substitution ofthe aspartic acid corresponding to position 20 of the mature human IL-2moiety with a threonine (D20T). In yet another preferred embodiment, theamino acid mutation is a substitution of the asparagine at position 88of the mature human IL-2 protein with an arginine (N88R). Fusionproteins of the invention can also include mutations at more than oneamino acid positions. In one embodiment, a fusion protein according tothe invention includes amino acid substitutions changing an asparagineto an arginine at position 88, a leucine to a threonine at position 85and an isoleucine to a threonine at position 86 of the mature human IL-2protein.

Mutations of amino acids at certain positions in the IL-2 moiety resultsin a differential effect that is greater than about 2-fold. It is usefulto mutate amino acids corresponding to positions K8, Q13, E15, H16, L19,D20, Q22, M23, N26, H79, L80, R81, D84, N88, I92, and E95 of the maturehuman IL-2 protein. Additional useful amino acid positions that can bemutated are L25, N31, L40, M46, K48, K49, D109, E110, A112, T113, V115,E116, N119, R120, I122, T123, Q126, S127, S130, and T131 of the maturehuman IL-2 protein. Preferred amino acid positions that are mutated infusion proteins of the invention include D20, N88, and Q126.

In one embodiment, one or more amino acid at the preferred positionslisted above are mutated in the fusion proteins. In a preferredembodiment, the amino acid asparagine at position 88 is substituted withan arginine (N88R). In another preferred embodiment, the amino acidaspartic acid at position 20 is substituted with a threonine (D20T). Inyet another preferred embodiment, the glutamine at position 126 issubstituted with an aspartic acid (Q126D). The various amino acidsubstitutions result in a selectivity in the activity of fusion proteinsof the invention for IL-2Rαβγ receptor bearing cells relative to IL-2Rβγreceptor bearing cells, which can be reflected in the fusion protein'saffinity for an IL-2Rβγ receptor relative to the fusion protein'saffinity for an IL-2Rαβγ receptor.

Fusion proteins with mutations at one or more amino acid positionsdescribed above have a differential effect that is greater than about2-fold. Preferably, the differential effect is between about 5-fold andabout 10-fold and more preferably between about 10-fold and about1000-fold.

In addition to mutating amino acids in the IL-2 moiety, amino acids inthe non-IL-2 moiety can also be mutated. In a preferred embodiment, thenon-IL-2 moiety is an antibody domain. The antibody domain can beselected from a variety of different immunoglobulin (Ig) antibodies,preferably IgG antibodies, including for example, IgG gamma 1, IgG gamma2 and IgG gamma 4 antibody domains, or any combination of these antibodydomains. As used herein, the terms “antibody” and “immunoglobulin” areunderstood to mean (i) an intact antibody (for example, a monoclonalantibody or polyclonal antibody), (ii) antigen binding portions thereof,including, for example, an Fab fragment, an Fab′ fragment, an (Fab′)₂fragment, an Fv fragment, a single chain antibody binding site, an sFv,(iii) bi-specific antibodies and antigen binding portions thereof, and(iv) multi-specific antibodies and antigen binding portions thereof. Inproteins of the invention, an immunoglobulin Fc region can include atleast one immunoglobulin constant heavy region, for example, animmunoglobulin constant heavy 2 (CH2) domain, an immunoglobulin constantheavy 3 (CH3) domain, and depending on the type of immunoglobulin usedto generate the Fc region, optionally an immunoglobulin constant heavy 4(CH4) domain, or a combination of the above. In particular embodiments,the immunoglobulin Fc region may lack an immunoglobulin constant heavy 1(CH1) domain. Although the immunoglobulin Fc regions may be based on anyimmunoglobulin class, for example, IgA, IgD, IgE, IgG, and IgM,immunoglobulin Fc regions based on IgG are preferred. An antibody moietyincluded in a fusion protein of the invention is preferably human, butmay be derived from a murine antibody, or any other mammalian ornon-mammalian immunoglobulin. It is contemplated that an Fc region usedin a fusion protein of the invention may be adapted to the specificapplication of the molecule. In one embodiment, the Fc region is derivedfrom an immunoglobulin γ1 isotype or a variant thereof. In anotherembodiment, the Fc region is derived from an immunoglobulin γ2 isotypeor a variant thereof. In further embodiments, the Fc region may bederived from an immunoglobulin γ3 isotype or a variant thereof. The Fcregion may comprise a hinge region that is derived from a differentimmunoglobulin isotype than the Fc region itself. For example, the Fcregion may be derived from an immunoglobulin γ2 isotype and include ahinge region derived from an immunoglobulin γ1 isotype or a variantthereof. In yet another preferred embodiment of the invention, the Fcregion is derived from an immunoglobulin γ4 isotype. Immunoglobulin γ4isotypes that have been modified to contain a hinge region derived froman immunoglobulin γ1 isotype or a variant thereof are particularlypreferred.

In one embodiment, fusion proteins of the invention comprise mutationsin the Ig moiety. A useful mutation is a mutation in the IgG gamma 1sequence QYNSTYR (SEQ ID NO: 1), changing the N to a Q; a particularlyuseful mutation is a mutation in the gamma 2 or 4 sequence QFNST (SEQ IDNO: 2), changing the dipeptide motif FN to AQ.

The invention also features DNA constructs encoding various fusionproteins of the invention. The fusion proteins of the invention areparticularly useful for treating cancer, viral infections and immunedisorders.

These and other objects, along with advantages and features of theinvention disclosed herein, will be made more apparent from thedescription, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the fusion of a cytokine to a second protein moietythat alters the natural binding characteristics of the cytokine. FIG. 1Adepicts the fusion partner to IL-2 as a dimeric molecule, such as anantibody or the Fc portion of an Fc-containing fusion protein, andtherefore two molecules of IL-2 are brought to the cell surface when theIL-2 moiety of the fusion protein interacts with its receptor. FIG. 1Billustrates a second mechanism that produces the same effect.

FIG. 2 shows typical pharmacokinetic profiles of the fusion proteinimmunocytokine huKS-IL2 (represented by triangles) and two variants,huKS-ala-IL2 (represented by circles) and huKS-ala-IL2(N88R)(represented by stars).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions that enhance thetherapeutic index of IL-2 fusion proteins and IL-2 immunocytokines inparticular. According to the invention, the therapeutic index of atherapeutic molecule is a measure of the ratio of the maximum tolerateddose of a molecule divided by the dose of maximal effectiveness for thatmolecule. The invention includes improved variants of IL-2immunocytokines that exhibit a significantly longer circulatinghalf-life compared to free IL-2. The invention also provides IL-2 fusionproteins, and in particular IL-2 immunocytokines, that exhibit aselective IL-2 response, reflected by reduced activation of cells withvarious effector functions by the fusion proteins of the invention,which is a leading cause of the toxic effects of IL-2. In addition, theinvention provides IL-2 fusion proteins with improved activity. An IL-2fusion protein of the invention includes changes at one or more aminoacid positions that alter the relative affinity of the IL-2 fusionprotein for different IL-2 receptors, resulting in altered biologicalproperties of the IL-2 fusion protein. The invention is useful to reduceor minimize any toxicity associated with IL-2 therapy. Regardless of theunderlying mechanism of any given IL-2 toxicity, such as VLS, thetoxicity results in part from the fact that IL-2 is administeredintravenously and therefore acts systemically within the body, eventhough the effect of IL-2 is desired at a specific site. This problem isexacerbated by the fact that a systemic administration of IL-2 requiresa much higher dose than a localized administration would, which in turnmay promote toxicities that would not be seen at lower doses. Theinvention provides IL-2 fusion proteins with reduced toxicity. Theinvention also provides methods for making IL-2 fusion proteins withreduced toxicity.

In general, the invention is useful for fusion proteins including anIL-2 moiety fused to a non-IL-2 moiety. According to the invention, anon-IL-2 moiety can be a synthetic or a natural protein or a portion orvariant (including species, allelic and mutant variants) thereof.Preferred non-IL-2 moieties include Fc and albumin moieties. Accordingto the invention, an IL-2 moiety can be a natural IL-2 molecule or aportion or variant (including species, allelic and mutant variants)thereof that retains at least one IL-2 activity or function (an IL-2moiety can be an IL-2 that is modified to have a different IL-2 receptorbinding affinity according to the invention).

According to the invention, cells respond to IL-2 through specific cellsurface receptors (IL-2R), which exist in two forms. The high affinityreceptor is heterotrimeric, consisting of α, β and γ subunits; theintermediate affinity receptor is heterodimeric, consisting of β and γsubunits. Binding constants of IL-2 for these two forms of IL-2R differby two orders of magnitude. Signal transduction is mediated on thecytoplasmic side of the receptor through interactions within the βγcomplex. Different cell types express the α, β and γ subunits in varyingamounts. For instance, activated T cells express all of the subunits toform the high affinity IL-2Rαβγ, whereas mature resting T cells and NKcells express the β and γ subunits to give the intermediate affinityIL-2Rβγ. Thus, cells require different levels of exposure to IL-2 forstimulation, and conversely, by regulating IL-2 activity within aspecific cellular context, the nature of an immune response can becontrolled.

Methods and compositions of the invention are particularly useful in thecontext of IL-2 fusion proteins such as IL-2 bearing immunocytokines.According to the invention, IL-2 bearing immunocytokines are syntheticmolecules that have been shown to significantly increase the efficacy ofIL-2 therapy by directly targeting IL-2 into a tumor microenvironment.Immunocytokines are fusion proteins consisting of an antibody moiety anda cytokine moiety, such as an IL-2 moiety. According to the invention,an antibody moiety can be a whole antibody or immunoglobulin or aportion or variant (including species, allelic and mutant variants)thereof that has a biological function such as antigen specific bindingaffinity. Similarly, a cytokine moiety of the invention can be a naturalcytokine or a portion or variant (including species, allelic and mutantvariants) thereof that retains at least some cytokine activity. Thebenefits of an immunocytokine therapy are readily apparent. For example,an antibody moiety of an immunocytokine recognizes a tumor-specificepitope and results in targeting the immunocytokine molecule to thetumor site. Therefore, high concentrations of IL-2 can be delivered intothe tumor microenvironment, thereby resulting in activation andproliferation of a variety of immune effector cells mentioned above,using a much lower dose of the immunocytokine than would be required forfree IL-2. In addition, the increased circulating half-life of animmunocytokine compared to free IL-2 contributes to the efficacy of theimmunocytokine. And finally, the natural effector functions of anantibody also may be exploited, for instance by activating antibodydependent cellular cytotoxicity (ADCC) in FcγRIII bearing NK cells.

An IL-2 immunocytokine has a greater efficacy relative to free IL-2.However, some characteristics of IL-2 immunocytokines may aggravatepotential side effects of the IL-2 molecule. Because of thesignificantly longer circulating half-life of IL-2 immunocytokines inthe bloodstream relative to free IL-2, the probability for IL-2 or otherportions of the fusion protein molecule to activate components generallypresent in the vasculature is increased. The same concern applies toother fusion proteins that contain IL-2 fused to another moiety such asFc or albumin, resulting in an extended half-life of IL-2 incirculation.

The invention provides altered IL-2 fusion proteins, such as IL-2 fusedto an intact antibody or to a portion of an antibody, or to albumin,with reduced toxicity compared to unaltered forms of such fusionproteins. The invention also provides fusion proteins with one or morealterations in the IL-2 and/or the non-IL-2 moieties that alter therelative activity of the fusion protein in cells expressing the α, β,and γ IL-2 receptor subunits compared to cells expressing the β and γIL-2 receptor subunits. The invention also provides for altered IL-2containing fusion proteins that exhibit an altered affinity towards theα, β, or γ subunit of the IL-2 receptor compared to unaltered forms ofsuch fusion proteins.

A number of IL-2-containing antibody fusion proteins exhibit IL-2activity that is quantitatively altered with respect to free IL-2, butis not qualitatively optimal for therapeutic applications. The inventionprovides modified forms of antibody-IL2 fusion proteins in which IL-2 orthe antibody, or both moieties, are altered to qualitatively improve theIL-2 activity for a given application.

The invention also provides strategies for determining the types ofmodifications that are particularly useful in designing modified fusionproteins for treatment of diseases.

FIG. 1 illustrates possible mechanisms by which a fusion protein maybind to a cell surface, such that the receptor-binding properties of amoiety within the fusion protein are altered. For example, FIG. 1Adepicts the fusion partner to IL-2 as a dimeric molecule. This increasesthe probability that the second IL-2 molecule interacts with itsreceptor, for example by decreasing the off-rate, which leads to a netincrease in binding. FIG. 1B illustrates a second mechanism thatproduces the same effect. In cells that bear both a receptor for IL-2and a receptor for the IL-2 fusion partner of the fusion protein (e.g.an Fc receptor for the Fc part of an Ig moiety) the receptor for thefusion partner (e.g. the Fc receptor) can engage the fusion protein andtether it at the cell surface where it now has an increased likelihoodto bind to an IL-2 receptor.

A Phase I/II trial of an antibody-cytokine fusion protein, termedhuKS-IL2, was recently completed. huKS-IL2 is a fusion proteinconsisting of the KS-1/4 antibody fused to the cytokine, interleukin-2.KS-1/4 recognizes the tumor cell surface antigen EpCAM (epithelial celladhesion molecule) and has the effect of concentrating IL-2 at the tumorsite. In the course of this trial, patient responses to treatment weremeasured. One patient who showed significant response to the therapyexperienced a clinical partial response followed by diseasestabilization and reduction in the use of pain medication. The patienthad already received prior standard treatments that had failed. Thepatient's life was extended significantly beyond what was expected inthe absence of such treatment.

Surprisingly, as a result of prior chemotherapy, this patient's T cellpopulation was essentially obliterated. This patient had much lower Tcell counts than all the other patients in the trial. Given that IL-2 isknown to activate T cells and, for example, is known to enhance thecytotoxicity of CD8(+) T cells toward tumor cells, the strong responseof this patient apparently lacking T cells was particularly unexpected.This observation prompted further study of novel antibody-IL-2 fusionproteins in which the IL-2 moiety might exhibit altered cellspecificity, resulting in an improvement in the therapeutic index ofIL-2 fusion proteins.

From the crystal structure of IL-2, sequence comparisons with relatedcytokines, and site-directed mutagenesis studies, much progress has beenmade in elucidating amino acids in IL-2 that come in contact withdifferent IL-2 receptor subunits and their consequence on biologicalactivity. For instance, the D20 residue, conserved in IL-2 acrossmammalian species, is a critical residue for binding the β subunit ofthe IL-2 receptor and various substitutions at this position havedistinct effects. For example, the variant IL-2(D20K) fails to bind toany IL-2R complex and is generally inactive, while variants IL-2(D20E)or IL-2(D20T) retain their biological activity. Amino acid positions R38and F42 are critical for binding the α subunit, and while mutations atthese sites diminish the interaction of IL-2 with the high affinityreceptor IL-2Rαβγ, it still binds to the intermediate affinity receptorIL-2Rβγ and thus some bioactivity is retained. N88 is another residuethat is involved in mediating interactions with the β subunit, and whilethe IL-2 (N88R) variant has greatly reduced affinity for theintermediate affinity receptor, its affinity for the high affinityreceptor is essentially unchanged. The N88R mutant of IL-2 is thereforestill able to activate T cells.

Binding affinity of fusion proteins of the invention for differentreceptors can be determined by a number of methods known in the artincluding, for example, a radioimmunoassay.

It is thus possible to perturb the IL-2 structure so that it displaysgreater affinity toward one IL-2 receptor complex compared with anotherIL-2 receptor complex by mutating a specific amino acid that contactsone of the receptor subunits, or by altering a combination of amino acidresidues. As a consequence, the molecule displays greater activity inone cell type versus another. According to the invention, it is possibleto manipulate the structure of IL-2 in the context of an Ig-IL2 fusionprotein to obtain the desired effect. Moreover, in some instances, theIg-IL2 variant fusion protein possesses different biologicalcharacteristics compared to the corresponding free IL-2 mutant protein.

It is furthermore possible, according to the invention, to manipulatethe IL-2 moiety in a fusion protein so that it displays an alteredaffinity toward one or more of the IL-2 receptor subunits (α, β, or γ)and results in an overall decrease in bioactivity of the fusion protein.Such variants are able to activate IL-2 responsive cells, but require ahigher concentration than free IL-2. Accordingly, when IL-2 fusionproteins are concentrated at a desired target site, for example by atargeting moiety, these variants have an improved therapeutic index.

The α receptor subunit of IL-2R appears to play a tethering function:this low-affinity receptor binds to IL-2 and keeps IL-2 close to thecell surface, so that the effective concentration in the neighborhood ofcell surface IL-2Rβ and IL-2Rγ receptor subunits is increased. Together,the α-subunit and the βγ-subunits of the IL-2 receptor create the highaffinity IL-2R complex. The invention is based, in part, on therecognition that IL-2 fusion proteins can engage in multiple anddistinct interactions with receptors on the cell surface. For example,in the case of fusion proteins containing an antibody moiety, theantibody moiety itself may promote binding of the fusion protein to thecell surface and furthermore, IL-2 may be present in multiple copies inthe fusion protein. As a result, IL-2 may be tethered to a cellexpressing only the β and γ subunits of IL-2R, and have an enhancedability to activate such a cell.

For example, a dimeric immunoglobulin (Ig) fused to IL-2 possesses twocopies of IL-2, such that the binding of one IL-2 moiety to its receptorenhances the probability of an interaction of the second IL-2 moietywith a receptor molecule on the same cell surface. The diagram in FIG.1A represents a possible configuration of an Ig-IL2 fusion protein on acell surface. The invention provides Ig-IL2 fusion proteins in which theIL-2 moiety is altered to reduce binding to an IL-2Rβγ receptor.

A second mechanism by which Ig-IL2 fusion proteins may have alteredbinding to the surface of certain immune cells is that the Fc receptoron a cell surface may bind to the Fc part of an Ig moiety and thustether the IL-2 to the surface of cells possessing both an Fc receptorand an IL-2 receptor (FIG. 1B). Such cells include NK cells, B cells,and macrophages. The invention provides Ig-IL2 fusion proteins in whichthe Ig moiety is altered to reduce binding to an Fc receptor. Theinvention further provides Ig-IL2 fusion proteins in which both theIg-moiety and the IL-2 moiety incorporate alterations of the naturedescribed above.

Based on the insight that Ig-IL2 fusion proteins may be artificiallytethered to cells bearing IL-2 receptor subunits, it is possible todesign variant fusion proteins in which the tethering moiety is altered.For example, it is useful to alter the Fc-receptor binding features ofan Ig-IL2 fusion protein. This may be done, for example, by mutatingknown amino acid contact sites within the Fc moiety or by removing theN-linked glycosylation sites, either by mutation or by enzymaticdigestion of the protein.

Similarly, according to the invention it is useful to introducemutations within the IL-2 moiety that have an effect on binding to IL-2receptor subunits. In particular, it is useful to mutate amino acids inIL-2 that come into contact with the β subunit of IL-2 receptor. Aparticularly useful type of mutation is one that reduces the energy ofbinding between IL-2 and IL-2Rβ, but does not sterically hinder thisinteraction. For example, mutation of a contact amino acid to an aminoacid with a smaller side chain is particularly useful. The effect ofsuch mutations is to reduce affinity of IL-2 for the β-γ form of IL-2receptor by a significant degree and also to reduce the activation ofthe signaling pathway mediated by these receptors, but to haverelatively little or no effect on binding to the α-β-γ form of the IL-2receptor or on the activity elicited by IL-2 in cells bearing such IL-2receptors. In a preferred embodiment of the invention, a mutationreduces the affinity for the β-γ form of the IL-2 receptor, but does noteliminate it.

Similarly, it is useful to introduce mutations in amino acids on thesurface of IL-2 that interact with the α subunit of IL-2 receptor. Aparticularly useful type of mutation is one that reduces the energy ofbinding between IL-2 and IL-2Rα, but does not sterically hinder thisinteraction. For example, mutation of a contact amino acid to an aminoacid with a smaller side chain is particularly useful. The effect ofsuch mutations is to reduce the affinity for the α-β-γ form of IL-2receptor to a significant extent, but to have relatively little or noeffect on binding to the β-γ form of the IL-2 receptor. In a preferredembodiment of the invention, a mutation reduces the affinity for theα-β-γ form of the IL-2 receptor, but does not eliminate it.

Similarly, it is also useful to introduce mutations in amino acids onthe surface of IL-2 that interact with the γ subunit of IL-2 receptor.As in the preceding cases, a particularly useful type of mutationreduces the energy of binding between IL-2 and IL-2Rγ, but does notsterically hinder this interaction. For example, mutation of a contactamino acid to an amino acid with a smaller side chain is particularlyuseful. The effect of such mutations is to reduce the affinity for theβ-γ form of IL-2 receptor to a significant extent, but to haverelatively little or no effect on binding to the α-β-γ form of the IL-2receptor. In a preferred embodiment of the invention, a mutation reducesthe affinity for the β-γ form of the IL-2 receptor, but does noteliminate it.

It is also useful to introduce a combination of amino acid mutationsinto IL-2 that interact with different surfaces of the IL-2 receptorsubunits. While each mutation independently may have little or no effecton binding of IL-2 to either the α-β-γ or the β-γ form of the IL-2receptor, the combination of mutations may achieve the desired reductionin affinity of IL-2 for its receptor or the bioactivity of IL-2.

According to the invention, mutations in other parts of IL-2 indirectlycontribute to alterations in the interaction of IL-2 with either the β-γform or the α-β-γ form of the IL-2 receptor, and thereby result in anIL-2 molecule with modulated activity. For instance, a mutation mayslightly alter the conformation of the molecule and alter its bindingproperties.

According to the invention, it is also useful to produce fusion proteinsthat contain mutations in the IL-2 moiety that modulate binding of theIL-2 moiety to an IL-2 receptor complex and also mutations in theantibody moiety. These fusion proteins may be particularly useful if itis desired to alter the interaction of the Ig-IL2 fusion protein withparticular Fc receptors.

A free IL-2 moiety can display different binding characteristics for anIL-2R complex than when the IL-2 moiety is fused to another proteinmoiety such as an Ig. One possible mechanism by which this occurs ispresented above. Another possible mechanism is that IL-2 is stericallyor conformationally constrained in the context of the immunocytokine andthat the particular constraint is reflected in the bindingcharacteristics of the IL-2 moiety towards the different IL-2 receptorcomplexes. It is therefore useful to introduce alterations in the fusionprotein that will modulate this constraint. For example, changes in thenon-IL-2 moiety are useful in modulating the activity of IL-2.

The usefulness of a particular IL-2 fusion protein, such as an Ig-IL2fusion or an IL-2 fusion protein containing Fc or albumin, for aparticular application, such as treatment of human disease, is tested inan appropriate cellular or animal model. When possible, testing in ananimal is preferred, because such testing comes closer to the fullcomplexity of the behavior of the immune system in a human disease. Forexample, a particular balance of certain cells may be optimal to fight adisease of interest, such as cancer or an infection with a bacterium,virus, or parasite. For example, a relatively high level of T cellactivity may be useful against a certain tumor type, while a relativelyhigh level of NK cell activity may be useful against a different tumortype.

Another feature of the invention is IL-2 fusion protein variants, suchas Ig-IL2 fusions or IL-2 fusions containing Fc or albumin, withsuperior toxicity profiles. For example, an Ig-IL2 fusion proteincontaining the mutation D20T shows reduced toxicity in animals such asmice as compared to corresponding Ig-IL2 fusion proteins with D atposition 20. In another example, an Ig-IL2 fusion protein containing themutation N88R or the combination of mutations L85T, I86T, N88R in theIL-2 moiety shows reduced toxicity in animals such as mice as comparedto corresponding Ig-IL2 fusion proteins with N at position 88. Inaddition, an antibody-IL2 fusion protein containing the mutation D20T orthe mutation N88R in the IL-2 moiety shows comparable potency to thecorresponding parental antibody-IL2 fusion protein when used to treat atumor that expresses an antigen target of the antibody.

The properties of the D20T variant of Ig-IL2 fusion proteins isparticularly surprising in light of the reported properties of the D20Tmutation in the free IL-2 protein. Specifically, the D20T mutation inthe free IL-2 protein does not display a difference relative to thewild-type IL-2 protein in its activity towards IL-2Rαβγ-bearing cells orIL2R-βγ-bearing cells (Shanafelt et al., PCT WO99/60128). However, anIg-IL2 fusion protein containing the D20T mutation has a drasticallyreduced potency in activation of IL2R-βγ-bearing cells, but hasessentially normal potency in activating IL-2Rαβγ-bearing cells.

Accordingly, mutation of several amino acids within the IL-2 moiety ofan Ig-IL2 fusion protein leads to reduced toxicity while havingrelatively little effect on the potency of the fusion protein in thetreatment of various diseases. For instance, the extent to which theaffinity of an IL-2 fusion protein variant for its receptors may bealtered is a function of how well the particular fusion protein isconcentrated at its intended target site. It is particularly useful tomutate one or more of the following amino acids within the IL-2 moiety:Lys8, Gln13, Glu15, His16, Leu19, Asp20, Gln22, Met23, Asn26, Arg38,Phe42, Lys43, Thr51, His79, Leu80, Arg81, Asp84, Asn 88, Val91, Ile92,and Glu95. It is also useful to mutate one or more of the followingamino acids within the IL-2 moiety: Leu25, Asn31, Leu40, Met46, Lys48,Lys49, Asp109, Glu110, Ala112, Thr113, Val115, Glu116, Asn119, Arg120,Ile122, Thr123, Gln126, Ser127, Ser130, and Thr131.

This invention discloses forms of an Ig moiety fused to IL-2, forexample antibody-IL2 fusions such as huKS-IL2 or dI-NHS76-IL2, in whichchanges in the Ig moiety fused to IL-2 affect the binding properties ofthe fusion protein to the IL-2R complex. These changes may be amino acidsubstitutions in the amino acid sequence of the heavy chain, or chemicalmodifications. Useful amino acid substitutions include those that affectthe glycosylation of the fusion protein or that directly affectinteraction with an Fc receptor. A particularly useful substitution maybe one that inhibits the glycosylation normally found at position N297(EU nomenclature) of the IgG heavy chain. Chemical and biochemicalmodifications include PEGylation of the molecule or treatment withN-glycanase to remove N-linked glycosyl chains. Without wishing to bebound by theory, one may envisage that specific changes in the antibodyportion of the molecule could affect the conformation of IL-2, forinstance by altering the rigidity of the antibody molecule. In the caseof huKS-IL2, these alterations may lead to a KS-IL2 molecule which nowshows an increased selectivity towards T cells in a cell based bioassay.

For antibody-IL2 fusion proteins it is often useful to select an Igmoiety that confers other desired properties to the molecule. Forexample, an IgG moiety of the gamma 1 subclass may be preferred tomaintain immunological effector functions such as ADCC. Alternatively,an IgG moiety of the gamma 2 or gamma 4 subclasses may be preferred, forexample to reduce FcR receptor interactions. When using IgG moieties ofsubclasses gamma 2 or gamma 4, inclusion of a hinge region derived fromgamma 1 is particularly preferred.

It is often useful to use the mutations and chemical or biochemicalmodifications of Ig-IL2 fusion proteins in combination with othermutations having distinct useful properties, such as the mutation of thelysine at the C-terminus of certain Fc regions to an alanine or anotherhydrophobic residue. For example, it is particularly useful to apply themodifications of the invention to the antibody fusion proteinhuKS-ala-IL2 or dI-NHS(76)-ala-IL2. It is also preferred to introducefurther mutations into the molecule that eliminate potential T-cellepitopes. It is particularly preferred that these mutations do notsubstantially alter the desired properties of the molecule.

This invention further discloses forms of an Ig moiety fused to IL-2,for example an antibody-IL2 fusion such as huKS-IL2, in which a specificalteration in the amino acid sequence of IL-2, for example IL2(D20T) orIL2(N88R) changes the binding properties of the fusion protein to theIL-2R complex. The amino acid sequence of mature human IL-2 protein isdepicted in SEQ ID NO: 3. The changes in binding properties arereflected in an increased selectivity towards T cells in a cell basedbioassay. The particular mutation influences the degree of selectivitytowards T cells. In addition, these changes result in a fusion molecule,for instance huKS-ala-IL2(D20T) or huKS-ala-IL2(N88R), with less toxicside effects when administered to mice systemically than, for instancehuKS-ala-IL2. Also, these changes lead to a fusion protein, for instancehuKS-ala-IL2(N88R), that is at least as efficacious as the normalhuKS-IL2 or huKS-ala-IL2 in tumor therapy in a number of mouse tumormodels.

Because the immunological responses required to clear a tumor aremanifold and also vary from tumor type to tumor type, it may not bedesirable to completely eliminate a functionality from the molecule whena molecule with reduced toxicity is used. For instance, in a mouse modelwhere pulmonary metastasis of colon carcinoma was induced, huKS-IL2 wasshown to effectively treat the cancer by a T cell mediated mechanism,which did not require NK cells, whereas in a mouse model forneuroblastoma, the elimination of the tumor by huKS-IL2 was shown torequire NK cells but not T cells. Therefore, there are cases where theselectivity profile may be more appropriately modulated to still allowan NK mediated response. In one embodiment of the invention, a moredesirable approach is to subtly alter the selectivity profile of themolecule such that a response involving multiple receptor types is stillachieved, most preferably at the sites where the molecule isconcentrated. For example, the invention provides alterations of anIg-IL2 fusion protein in which the selectivity for the IL-2Rαβγ,relative to IL-2Rβγ, is enhanced 2- to 10-fold, 10- to 100-fold, 100- to1000-fold, or more than 1000-fold, relative to a correspondingunmodified Ig-IL2 fusion protein.

Another object of the invention is to provide for optimal uses of Ig-IL2fusion proteins with reduced toxicity for the treatment of cancer orinfectious disease. While altered selectivity may lead to reducedvascular toxicity, it may not lead to optimal increases in thetherapeutic index upon increasing the dose of the fusion protein. Forexample, these increases in dose may lead to an induction of negativeregulatory mechanisms that regulate immune responses. It may thereforebe useful to use treatment modalities that combine low-toxicity Ig-IL2fusion proteins with agents that decrease such effects.

One recently identified potent inhibitor of cellular immune responses isa class of CD4⁺CD25⁺ regulatory T cells that express the high affinityIL-2R (for a review, see Maloy and Powrie, (2001) Nature Immunol.2:816). According to the invention, increased doses of low-toxicityIg-IL2 fusion proteins may additionally activate these cells. Uponstimulation, these cells up-regulate CTLA-4 on their cell surface, whichengage cell surface molecules B7-1 and B7-2 on immune cells and in turnelicit a potent negative signal (Takahashi et al., (2000) J. Exp. Med.192: 303). Thus, inhibitors of these processes could be useful incombination therapy with fusion proteins of the invention. In oneembodiment, antibodies neutralizing CTLA-4 and its effects can be used.In another embodiment, other proteins with similar activity can be used,such as soluble B7 receptors and their fusion proteins (e.g. B7-Ig).Further embodiments include the use of antibodies that kill or inhibitthese regulatory T cells themselves such as anti-CD4 and anti-CD25. In apreferred embodiment, the latter are administered sequentially ratherthan simultaneously.

According to the invention, another useful mechanism involvesoverstimulation of cyclo-oxygenase 2 (COX-2) leading to the productionof prostaglandins, which are known to inhibit immune responses (see PCTUS99/08376). Therefore, a further embodiment combines the use of thelow-toxicity Ig-IL2 molecules with COX-2 inhibitors such asIndomethacin, or the more specific inhibitors Celecoxib (Pfizer) andRofecoxib (Merck&Co). It is understood that still other immunemechanisms might be activated by increasing doses of low-toxicity Ig-IL2fusion proteins and that combination therapies may be devised to addressthese mechanisms. In addition, low doses of certain cytotoxic drugs,such as cyclophosphamide, which have immune potentiating effects in vivomay be useful therapeutic agents to include in a combination therapy.

Fusions of albumin have been developed with the purpose of generatingtherapeutic fusion proteins with enhanced serum half-lives. For example,Yeh et al. (Yeh P, et al. Proc Natl Acad Sci USA. [1992] 89:1904–8.)constructed an albumin-CD4 fusion protein that had a much longer serumhalf-life than the corresponding CD4 moiety alone.

It is useful to construct fusions of albumin to IL-2, erythropoietin,interferon-alpha, and other ligands. These fusion proteins have longerserum half-lives than the corresponding ligand alone. Such fusions maybe constructed, in the N- to C-terminal direction, as ligand-albuminfusions or albumin-ligand fusions, using standard genetic engineeringand protein expression techniques. Alternatively, albumin and a ligandmay be joined by chemical conjugation.

However, albumin-ligand fusion proteins often have undesirableproperties. Without wishing to be bound by theory, one reason for whyalbumin-ligand fusion proteins may have undesirable properties is thefact that there are receptors for albumin on vascular endothelial cells(Tiruppathi et al. Proc Natl Acad Sci USA. [1996] 93:250–4). As aresult, the effects of a ligand on vascular endothelial cells may beenhanced.

For example, an albumin-IL2 fusion protein has an enhanced serumhalf-life, but also causes enhanced vascular leak. Without wishing to bebound by theory, it is noted that activation of IL-2 mediated responsesin the vasculature is increased because of binding of the fusion proteinto albumin receptors present on endothelial cells of the vasculature.Binding of albumin-IL2 fusion proteins to cells that have receptors bothfor albumin and IL-2 is enhanced by a mechanism analogous to that shownin FIG. 1 b for the enhancement of binding of an Ig-ligand fusionprotein to a cell surface.

To reduce the vascular leak caused by albumin-IL2, it is useful tointroduce mutations into the TL-2 moiety that specifically reduce IL-2'saffinity for IL-2Rβγ receptors. For example, an albumin-IL2(N88R) oralbumin-IL2(D20T) fusion protein is constructed and subsequently foundto have reduced toxicity and an enhanced therapeutic index for a diseasemodel in an animal such as a mouse.

Molecules of the present invention are useful for the treatment ofmalignancies and tumors, particularly treatment of solid tumors.Examples of tumors that can be treated according to the invention aretumors of epithelial origin such as those present in, but not limitedto, ovarian cancer, prostate cancer, stomach cancer, hepatic cancer,bladder, head and neck cancer. Equally, according to the invention,malignancies and tumors of neuroectodermal origin are suitablecandidates for treatment, such as, but not limited to, melanoma, smallcell lung carcinoma, soft tissue sarcomas and neuroblastomas.

According to the invention, it is useful for the therapeutic agent to betargeted to the tumor site or the site of the malignancy or metastasis.Ig-fusion proteins containing antibodies directed toward antigenspreferentially presented by tumors or malignant cells are particularlyuseful. For example, fusion proteins containing an antibody moiety withspecificity for EpCAM (eg KS1/4), or embryonic Fibronectin (eg. BC1), orCEA, or chromatin complexes (eg. NHS76), or GD2 (eg 14.18), or CD19, orCD20, or CD52, or HER2/neu/c-erbB-2, or MUC-1, or PSMA are particularlyuseful. In addition, antibodies directed to various viral antigens areparticularly useful.

EXAMPLES Example 1

Construction of Ig-IL2 Fusion Genes with Codon Substitutions in the IL-2Coding Sequence or in the Antibody Coding Sequence:

An expression vector for immunocytokines was described in Gillies etal., (1998) J. Immunol. 160:6195–6203. Several modifications in thenucleotide sequence enabled the addition of coding sequences to the 3′end of the human γ-1 gene. In the human γ-1 gene encoding the heavychain, the XmaI restriction site located 280 bp upstream of thetranslation stop codon was destroyed by introducing a silent mutation(TCC to TCA). Another silent mutation (TCT to TCC) was introduced to theSer codon three residues upstream of the C-terminal lysine of the heavychain to create the sequence TCC CCG GGT AAA (SEQ ID NO. 4), whichcontains a new XmaI site [Lo et al., (1998) Protein Engineering11:495–500].

The IL-2 cDNA was constructed by chemical synthesis and it contains anew and unique PvuII restriction site [Gillies et al., (1992) Proc.Natl. Acad. Sci. 89:1428–1432]. Both the XmaI and PvuII sites are uniquein the expression vector, and they facilitated construction ofantibody-IL2 variants, including the following.

1) huKS-ala-IL2. The construction of huKS-ala-IL2 has been describedpreviously (e.g. WO01/58957). The resulting protein contains an aminoacid substitution at the junction between the Ig heavy chain constantregion and mature huIL-2. The junction normally has the sequenceSPGK-APT (SEQ ID NO: 5) in which —SPGK- is the C-terminus of the heavychain and —APT- the N-terminus of the mature IL-2 protein. InhuKS-ala-IL2 a K to A substitution was introduced (referred to asposition K[−1]) and the junction now has the sequence SPGA-APT (SEQ IDNO: 6). As a consequence the serum half-life of this protein is improved(see Example 5).

2) dI-KS-ala-IL2. This KS-IL2 fusion protein contains substitutions inKS-ala-IL2 to generate a version of the fusion protein in whichpotential T-cell epitopes have been eliminated (described in co-pendingpatent applications U.S. Ser. Nos. 10/112,582 and 10/138,727, the entiredisclosures of which are incorporated by reference herein).

The constant region of the Ig portion of the fusion proteins of theinvention may be selected from the constant region normally associatedwith the variable region, or a different constant region resulting in afusion protein with the Ig portion including variable and constantregions from different subclasses of IgG molecules or different species.For example, the gamma4 constant region of IgG (SEQ ID NO: 7) may beused instead of gamma1 constant region (SEQ ID NO: 8). The alterationhas the advantage that the gamma4 chain can result in a longer serumhalf-life. Accordingly, IgG gamma2 constant region (SEQ ID NO: 9) mayalso be used instead of IgG gamma1 constant region (SEQ ID NO: 8). Inaddition, the hinge region derived from IgG gamma1 (SEQ ID NO: 10) mayreplace the hinge region normally occurring in IgG gamma2 (SEQ ID NO:11) or IgG gamma4 constant region (SEQ ID NO: 12). The Ig component ofthe fusion protein may also include mutations in the constant regionsuch that the IgG has reduced binding affinity for at least one ofFcγRI, FcγRII or FcγRIII. The fusion proteins of the invention mayinclude mutations in the IgG constant regions to remove potentialglycosylation sites and T-cell epitopes. For example, the variousconstant regions may include alterations in the C-terminal part of theconstant regions to remove potential T-cell epitopes. For example,potential T-cell epitopes in the C-terminal part of various constantregions of IgG molecules are removed by changing the amino acid sequenceKSLSLSPGK (SEQ ID NO: 13) in IgG gamma1 and IgG gamma 2 constant regionsand amino acid sequence KSLSLSLGK (SEQ ID NO: 14) in IgG gamma4 constantregion to amino acid sequence KSATATPGA (SEQ ID NO: 15).

3) huKS-ala-IL2(N88R). This huKS-IL2 variant contains the same aminoacid substitution at the junction between the Ig heavy chain constantregion and mature huIL-2 as described above (K[−1]A, created by thecodon change AAA to GCC), and in addition it contains a substitution atposition N88 in the sequence of mature huIL-2 in favor of R (created bycodon change aAT to aGG). A further alteration was introduced into thenucleotide sequence of huIL-2 to eliminate an existing restriction sitefor Bam HI by introducing a silent mutation (amino acid position G98,the codon was switched from ggA tcc to ggC tcc).

A PCR-based mutagenesis strategy was used in the construction ofhuKS-ala-IL2(N88R). Two overlapping PCR fragments that span the codingsequence of the mature huIL2 were generated using huIL2 in a Bluescriptvector (Stratagene) as a template. The upstream PCR fragment containedthe nucleotide changes encoding K[−1]A and N88R by incorporating thesemutations into the sense and antisense primers respectively. Thesechanges are indicated by the bold nucleotides in the primer sequences.The sense primer sequence was: 5′CCCCGGGTGCCGCCCCAACTTCAAGTTCTACA3′ (SEQID NO: 16); the antisense primer sequence was: 5′AGCCCTTTAGTTCCAGAACTATTACGTTGATCCTGCTGATTAAGTCCCTAGGT 3′. (SEQ ID NO:17). The underlined nucleotide represents a change that destroys the BamHI site. The second, downstream PCR fragment contained a 20 nucleotideoverlap region with the upstream PCR fragment and the remaining IL2sequence. The sense primer used in this reaction was 5′AGTTCTGGAACTAAAGGGCTCCGAAACAACATTCATGTGT (SEQ ID NO: 18). Again, theunderlined nucleotide denotes the silent mutation that destroys the BamHI site. The antisense primer used was the standard M13 reverse primerthat anneals to a sequence in the pBluescript vector. These overlappingPCR fragments were used in a reaction with the primer in SEQ ID 16 andan M13 reverse primer to generate the final PCR product, which wassubsequently inserted into a TA vector (Invitrogen).

The sequence of the inserted fragment was verified, and a 442 bp XmaI/Xho I fragment containing the modified IL2 sequence (from plasmidTA-IL2(N88R)) was used to replace the wild-type huIL-2 sequence in theparental immunocytokine expression plasmid (encoding huKS-IL2). Theresultant immunocytokine expression plasmid encoding huKS-ala-IL2(N88R)was verified by restriction mapping and sequencing.

4) huKS M1-IL2(TTSR (SEQ ID NO: 19)). The immunocytokine variant huKSM1-IL2 was constructed by standard recombinant DNA techniques (anddescribed e.g. in co-pending patent application U.S. Ser. No.10/112,582, the entire disclosure of which is incorporated by referenceherein). It contains multiple amino acid substitutions in the antibody—IL-2 junction region of the fusion protein, which eliminate potentialT-cell epitopes and results in a less immunogenic protein. The sequencewas changed from KSLSLSPGA-APT (SEQ ID NO: 20) to KSATATPGA-APT (SEQ IDNO: 21) (the dash denotes the Ig/IL-2 junction site and substitutedamino acids are underlined) and is denoted as “M1”. Also incorporated inthis variant is the K to A change at the last amino acid before thejunction that has been shown to increase serum half-life of theimmunocytokine.

huKS M1-IL2(TTSR) contains further amino acid substitutions located inthe IL-2 portion of the immunocytokine. To eliminate potential T-cellepitopes created by the substitution of N88R described above, thesequence is changed from —DLISNI-(SEQ ID NO: 22) of the natural huIL-2to —DTTSRI-(SEQ ID NO: 23).

A PCR based mutagenesis approach was used to introduce the changes intothe nucleotide sequence of the huIL-2 gene, by incorporating themutations into the sense primer. The sequence TTxR was created by codonchanges ACC, ACC and AGG respectively. A mutagenized 197 bp PCR fragmentencompassing the 3′ end of the hu IL-2 sequence was generated from thetemplate immunocytokine expression plasmid encoding huKS-ala-IL2(N88R)using a sense primer of the sequence 5′ACTTAAGACCTAGGGACACCACCAGCAGGATCAACGTAATAGT3′ (SEQ ID NO: 24) and anantisense primer of the sequence 5′ ATCATGTCTGGATCCCTC3′ (SEQ ID NO:25). The PCR fragment was cloned into a TA vector and the sequenceverified. To regenerate the complete IL-2 sequence this fragment wasligated as a Afl II/Xho I restriction digest to a 2 kb Hind III/ Afl IIfragment obtained from immunocytokine expression plasmid encodinghuKS-ala-IL2(N88R) and inserted into a Hind III/Xho I restrictedpBluescript vector. The mutagenized IL-2 gene was then exchanged inplace of the natural huIL-2 sequence in an immunocytokine expressionplasmid encoding for KS M1-IL2 in a three-way ligation.

5) huKS(N to Q)-IL2. An immunocytokine expression plasmid encodinghuKS(N to Q)-IL2 was constructed using standard recombinant DNAtechniques. huKS(N to Q)-IL2 contains an amino acid substitution in theCH2 domain of the antibody Fc gamma 1 constant region that eliminatesN-linked glycosylation. The amino acid sequence is changed from QYNSTYR(SEQ ID NO: 1) to QYQSTYR (SEQ ID NO: 26), with the substituted aminoacid indicated in bold. Similarly, fusion proteins including gamma 2 andgamma 4 constant regions were constructed that contain mutations thatchange the amino acid sequence QFNST (SEQ ID NO: 2) to QAQST (SEQ ID NO:27), thereby additionally eliminating a potential T cell epitope.

Example 2

Chemical or Enzymatic Modifications of an Ig-IL2 Fusion Protein Leadingto Modified Receptor Specificity:

This example describes biochemical manipulations of the immunocytokineused to generate a PEGylated huKS-IL2 or to a deglycosylated huKS-IL2,and variants thereof. The same methods can be applied to other IL-2fusion proteins, such as the immunocytokine 14.18-IL2 oralbumin-cytokine fusions. These variants were used in a subsequentexample to investigate their effect on the proliferative response ofvarious cell lines in a cell based bioassay (Table 1) or on thepharmacokinetic properties of the molecule.

PEGylation of huKS-IL2. PEG (20,000) was covalently attached to theprotein via amine groups present on the protein. For this purpose areactive derivative of PEG containing a succinimide linker(mPEG-Succinimidyl Propionate, termed “SPA-PEG” below) was employed.huKS-IL2 was extensively dialyzed in an amine-free buffer composed of 50mM Sodium Phosphate (pH 7.5), 0.05% Tween 80, and concentrated. ExcessSPA-PEG was combined with huKS-IL2 at a molar ratio of either 5:1 or10:1. Immediately before use, a 5 mM SPA-PEG stock solution was preparedin deionized water. An appropriate volume of the SPA-PEG solution wascombined with huKS-IL2 and the reaction was incubated on a rockingplatform for 30 to 40 minutes at room temperature. A 5 to 10 molarexcess of glycine was added to quench the reaction, and the reactionproducts were purified by size exclusion chromatography. A Superdex 200column, equilibrated in 50 mM HEPES and 150 mM NaCl, was loaded with thereaction sample and eluting fractions containing the PEGylated proteinwere pooled and concentrated.

N-Glycanase treatment of huKS-IL2. huKS-IL2 (1.5 mg) was incubated with30 mU PNGaseF (New England Biolabs) overnight at 37° C. The reactionproduct was purified by passage over a ProteinA-Sepharose column andelution of the bound huKS-IL2 at pH 3. The eluate was neutralized andconcentrated in a spin column in a buffer of PBS and 0.05% Tween 80.Deglycosylation of huKS-IL2 was verified be size exclusionchromatography and on a urea gel.

Example 3

Expression and Purification of Ig-IL2 and Ig-IL2 Variants

The general procedure described here for huKS-ala-IL2(N88R) may be usedfor a wide variety of Ig-cytokine fusion proteins, including Ig-fusionsto mutant cytokines. To obtain stably transfected clones which expresshuKS-ala-IL2(N88R), DNA of the immunocytokine expression plasmidencoding huKS-ala-IL2(N88R) was introduced into the mouse myeloma NS/0cells by electroporation. NS/0 cells were grown in Dulbecco's modifiedEagle's medium supplemented with 10% heat-inactivated fetal bovineserum, 2 mM glutamine and penicillin/streptomycin. About 5×10⁶ cellswere washed once with PBS and resuspended in 0.5 ml PBS. 10 μg oflinearized plasmid DNA were then incubated with the cells in a GenePulser Cuvette (0.4 cm electrode gap, BioRad) on ice for 10 min.Electroporation was performed using a Gene Pulser (BioRad, Hercules,Calif.) with settings at 0.25 V and 500 μF. Cells were allowed torecover for 10 min on ice, after which they were resuspended in growthmedium and plated onto two 96 well plates. Stably transfected cloneswere selected by growth in the presence of 100 nM methotrexate (MTX),which was added to the growth medium two days post-transfection. Thecells were fed every 3 days for two to three more times, andMTX-resistant clones appeared in 2 to 3 weeks. Supernatants from cloneswere assayed by anti-Fc ELISA to identify high producers. High producingclones were isolated and propagated in growth medium containing 100 nMMTX.

The immunocytokine was purified from the tissue culture supernatant byProtein A affinity column chromatography. For huKS-ala-IL2(N88R), arecombinant Protein A (rPA) Agarose column was pre-equilibrated with tenvolumes of running buffer, such as 100 mM Arginine, 5 mM Citrate, 0.01%Tween 80 pH 5.6, and the column was loaded with filtered cell culturesupernatant containing huKS-ala-IL2(N88R) at 16 ml/min to a binding ofapproximately 40 mg/ml of rPA resin. The column was washed extensivelywith the same buffer and finally the immunocytokine was eluted in 50 mMglycine at pH 3. Peak fractions were collected and pH was adjusted toneutral with 1 N NaOH.

Example 4

Activity of Ig-IL2 Variants in Bioassays.

For cell based bioassays, cell lines that depend on IL-2 for growth wereutilized and the activity of Ig-fusion proteins, for example huKS-IL2and huKS-IL2 variants, was assessed by proliferation of these cells. Forinstance, CTLL-2 (ATCC# TIB-214; Matesanz and Alcina, 1996) and TF-1β(Farner et al., [1995] Blood 86:4568–4578) were used to follow a T cellresponse and an NK cell-like response, respectively. CTLL-2 is a murineT lymphoblast cell line that expresses the high affinity IL-2Rαβγ, andTF-1β is a human cell line derived from immature precursor erythroidcells that express the intermediate affinity IL-2Rβγ. Another usefulcell line for these assays is, for example, the cell line derived fromhuman adult T cell lymphoma Kit-225 (K6) (Uchida et al., [1987] Blood70:1069–1072). When paired with cell line TF-1β, the activity of thefusion proteins is evaluated in a pair of cell lines harboring receptorsof the same mammalian species. These assays may also be performed withcell populations derived from human PBMCs (Peripheral Blood MononuclearCells), either to isolate NK-cells, which bear IL-2Rβγ, or to produceactivated T cells, which express IL-2Rαβγ. Techniques to isolate thesecell populations from hu PBMCs are known to those of ordinary skill inthe art. For example, T cells, or PHA-blasts, are obtained by incubatingPBMCs for three days in 10 microgram/ml of phytohemagglutinin (PHA-P;L9017, Sigma, St. Louis). Resting NK cells are commonly obtained by anegative selection protocol, for instance using an NK-cell isolation kit(Miltenyi Biotec, Auburn, Calif.) for human cells. To correlate theactivity of these fusion proteins with results obtained from mouse tumormodels, it is also useful to perform these assays on cell populationsobtained from the mouse expressing one or the other IL-2 receptorcomplex. For example, an NK cell population may be obtained from spleensof recombinant-deficient (SCID) Balb/C mice using a SPINSEP™ murineNK-cell enrichment kit (Stemcell Technologies Inc, Vancouver, BC,Canada). The purity of any of these enriched populations can be assessedby FACS analysis.

Briefly, washed cells were plated at a density of 10,000 cells/well in a96 well microtiter plate and incubated in cell medium supplemented with,for example, purified huKS-IL2 or huKS-IL2 variants. In addition, wildtype huIL-2 protein, obtained from R&D Systems (Minneapolis, Minn.) wasassayed as a standard. The added protein was prepared as a dilutionseries over a roughly 1000-fold concentration range between 0.45 ng/mland 420 ng/ml (normalized with respect to molar equivalents of IL2).After 32 hours, 0.3 μCi of [methyl-3H]thymidine (Dupont-NEN-027) wasadded to each well and cells were incubated an additional 16 hours.Cells were then harvested and lysed onto glass filters. 3H-thymidineincorporated into DNA was measured in a scintillation counter.

An ED50 value for each huKS-IL2 protein variant with respect to cellproliferation was obtained from plotting a dose response curve andidentifying the protein concentration that resulted in half-maximalresponse. The selectivity of the response was expressed as a ratio ofED50 values for example, ED50 [TF1-β]/ED50 [CTLL-2]. Thus, a high ED50ratio indicated that a relatively higher dose of the protein wasrequired to elicit a TF-1β cell response as compared to a CTLL-2 cellresponse. The ratio of the ED50 values of the huKS-IL2 variants wascompared to free huIL-2 and the parental huKS-IL2 proteins. Thisnormalized value is a measure of the differential effect. A value largerthan the one obtained for the reference protein indicated a shift inselectivity toward CTLL-2 cells. In some cases it may be preferable toobtain ED50 ratios with cell lines that originate from the same species,so that IL-2 activities are not additionally influenced by cross-speciesdifferences in their interaction with the receptors. The followingexample uses murine CTLL-2 and human TF-1β cells to calculate ED50ratios with Ig-IL2 fusion proteins and free IL-2, and representativeresults from such an experiment are shown in Table 1.

TABLE 1 Protein ED50 Ratio IL-2 0.81 HuKS-IL2 0.11 HuKS-ala-IL2 0.17KS(NtoQ)-IL2 0.72 HuKS-ala-IL2(N88R) 2300 KS-IL2(TTSR) >6 HuKS-IL2PEGylated 1.99 HuKS-IL2 + Glycanase 0.45 14.18-IL2 0.07 14.18-IL2PEGylated 1.34 14.18-IL2 + Glycanase 0.21

In this example, compared with the ED50 ratio obtained with free IL-2(0.81), an approximately 5-fold lower ED50 ratio was obtained withhuKS-IL2 (0.17). This indicated that the fusion protein was shifted inits selectivity profile, displaying a greater selectivity towards TF-1βcells. A different antibody/IL-2 combination, 14.18-IL2, also was moreselective for TF1-β than IL-2 alone (ED50 ratio of 0.07), indicatingthat this effect was not limited to a specific antibody contained in theantibody-IL2 fusion protein, and the reduced activity of human Ig-IL2fusion proteins towards murine high affinity receptor bearing cellsrelative to huIL-2 may reflect a general feature of the Ig-IL2 fusionproteins.

Other variants had an altered ED50 ratio such that a CTLL-2 cellresponse was favored. A dramatic effect was seen withhuKS-ala-IL2(N88R), for which the ED50 ratio was greater than 2000,reflecting that TF-1β cell proliferation, mediated in these cells by theintermediate affinity receptor, was barely detectable. Thus, whilehuKS-ala-IL2(N88R) activated signaling of cells with IL-2Rαβγ, it didnot significantly activate cells with IL-2Rβγ. The activity ofhuKS-ala-IL2(N88R) could also be assayed on purified murine NK cellsexpressing the murine IL-2Rβγ complex; in contrast to what was reportedfor the free human IL2(N88R) protein—which indicated that theselectivity was virtually lost when mouse T and NK cells were examined(see Wetzel et al., ASCO 2001 Meeting Abstract)—the ED50 value forhuKS-ala-IL2(N88R) in the mouse NK cells was similar to that observedwith TF-1β cells.

Subtle shifts in the selectivity of the response towards CTLL-2 cellswere observed in Ig-IL2 variants with alterations that affectglycosylation of the antibody portion of the fusion protein.Specifically, KS(NtoQ)-IL2, which lacks a glycosylation site in the Fcportion of the antibody, displayed a 3-fold increase in ED50 Ratio(0.72) relative to huKS-IL2, whereas N-Glycanase treated huKS-IL2displayed a 2-fold increase (ED50 ratio of 0.45) relative to huKS-IL2.Likewise, N-Glycanase treatment of IL-2 fused to a different antibodymolecule lead to a similar result; for instance, N-Glycanase treated14.18-IL2 gave a 3-fold increase in the ED50 ratio as compared tountreated 14.18-IL2. These results indicated that certain alterations inthe antibody portion of the molecule itself affect the binding andactivation properties of an IL-2 molecule fused to it.

PEGylation of the fusion protein also altered its selectivity profile.Again, a shift towards CTLL-2 stimulatory activity was observed. ForhuKS-IL2, a PEGylated variant resulted in a 9-fold increase inselectivity in favor of CTLL-2 cells (ED50 ratio of 1.99), and for14.18-IL2 a 20-fold increase was induced by PEGylation (ED50 ratio of1.34).

In some instances, these shifts in selectivity for a given protein mayalso reflect the particular combination of cell types employed in theassays, as illustrated in representative results shown in Table 2. Forexample, when KS-IL2, KS-ala-IL2 and IL-2 were compared using the humanIL-2Rαβγ bearing cell line Kit 225 instead of murine CTLL-2, thepatterns of shift in selectivity was not maintained. Particularly withregards to Kit 225 cells, these three proteins exhibited essentiallyidentical activity. Mostly however, the trends in the selectivityresponse of Ig-IL2 variants between TF-1β cells and Kit-225 cells werefound to be similar to those established with TF-1β cells and CTLL-2cells, including the effect of deglycosylation of the Fc-moiety of aIg-IL2 fusion protein (see representative results in Table 2 below andExample 10).

TABLE 2 ED50 Ratio Protein TF-1β/Kit-225 IL-2 2.8 HuKS-IL2 4HuKS-ala-IL2 10.4 KS-ala-IL2(N88R) 52,000

In addition, it was found that Kit-225 cells were more sensitive to IL-2and IL-2 fusion proteins and variants thereof than CTLL-2 cells. Forexample, the ED50 value for huKS-ala-IL2 was 0.08 in Kit-225 cells and5.0 in CTLL-2 cells, and for KS-ala-IL2(N88R) it was 0.13 in Kit 225cells and 3 in CTLL-2 cells, indicating an approximately 10–50 foldincrease in sensitivity of Kit 225 cells in these assays. Thus the valueof the ED50 ratio for a given protein is dependent on the particularcombination of cell types employed.

Example 5

Pharmacokinetics of IL-2 Fusion Proteins with Modified Receptor BindingCharacteristics

The pharmacokinetic (PK) profile of huKS-ala-IL2(N88R) was compared tothe profile of huKS-ala-IL2 and huKS-IL2. For each protein, three 6–8week old mice were used. Twenty five μg of the fusion proteins, dilutedto 125 μg/ml in PBS, were injected in the tail vein of mice, and 50 μlblood samples were obtained by retro-orbital bleeding immediately afterinjection (0 hrs) and at 0.5, 1, 2, 4, 8, and 24 hrs post injection.Blood samples were collected in heparin-coated tubes to prevent bloodclotting, and immunocytokine levels in the post-cellular plasmasupernatant were measured in an ELISA assay. The procedure of the ELISAassay used for pharmacokinetic studies has been previously described(WO01/58957). This assay measured the presence of an intactimmunocytokine. Capture of the immunocytokine from plasma was carriedout on EpCAM-coated plates and the detection was performed with anHRP-conjugated antibody directed against IL-2. It had been shownpreviously that the huKS-IL2 variant with a K to A substitution in thejunction, huKS-ala-IL2, had a dramatic improvement in circulatinghalf-life as compared to huKS-IL2 (WO01/58957). In fact, the circulatinghalf-life of huKS-ala-IL2(N88R) was found to be similarly improved,indicating that the N88R alteration in the IL-2 portion of the moleculehad no substantial effect on the pharmacokinetics. Results of arepresentative experiment are shown in FIG. 2. FIG. 2 illustrates a timecourse of the concentration of the immunocytokine present in the serum(expressed as a percentage of the protein concentration remaining in theserum relative to the starting concentration present immediately afterintravenous administration) over 24 hours. Protein concentrations aredetermined in an ELISA assay in which the immunocytokine is captured byits antibody moiety and detected by its cytokine moiety. X-axis=time tin hours; Y-axis=log(% of remaining protein concentration).

Example 6

Toxicity of Ig-IL2 Fusion Proteins with Modified Receptor BindingCharacteristics in a Mammal

The relative toxicity of the KS-IL2 variants huKS-IL2, huKS-ala-IL2, andhuKS-ala-IL2(N88R) in mice was examined. As was shown in Example 5,huKS-ala-IL2 and huKS-ala-IL2(N88R) have substantially increased PK whencompared to huKS-IL2. Nonetheless, for comparison purposes, an identicaldosing schedule was used for the different molecules despite thedifference in PK. While a longer serum half-life is likely to increasethe efficacy of a therapeutic it may also lead to increased toxicity.Yet this example shows that, while huKS-ala-IL2 had increased toxicitycompared to huKS-IL2 (because of a longer circulating half-life),huKS-ala-IL2(N88R) had decreased toxicity compared to huKS-IL2 despite alonger circulating half-life.

Balb/C mice (3 animals per experimental condition) were given dailyintravenous injections of one of three proteins for five consecutivedays. The fusion proteins were diluted into 200 μl of PBS and wereadministered at the following dosage: huKS-IL2 and huKS-ala-IL2 at 25,50, or 75 μg per mouse, and huKS-ala-IL2(N88R) at 50, 75, or 100 μg permouse. A control group received intravenous injections of PBS. Survivalof the mice was monitored daily and the effect on mouse survival wasexamined. Mice survived administration of all doses of huKS-IL2.huKS-ala-IL2, however, was more toxic. While the mice tolerated a doseof 25 μg of huKS-ala-IL2, all 3 mice died on day 6 at a dose of 50 μg,and at a dose of 75 μg, two mice had died at day 4.5, and the thirdmouse at day 5. huKS-ala-IL2(N88R), on the other hand, was welltolerated at all doses, including 100 μg. Indeed, huKS-ala-IL2(N88R) wasalso administered at a dose of 200 μg per mouse, and the mice survived.Thus, huKS-ala-IL2(N88R) was significantly less toxic than huKS-ala-IL2.

Mice that had died during the course of the treatment with huKS-ala-IL2were dissected and their organs evaluated. All organs, including lung,spleen, liver, stomach, and kidney were grossly distended, indicative ofextensive vascular leakage. Organs of animals treated with varianthuKS-ala-IL2(N88R) were also evaluated. Mice were treated as describedabove, and it was found that organ weights fromhuKS-ala-IL2(N88R)-treated animals were generally similar to those ofcontrol animals, particularly for the lungs and liver. Without wishingto be being bound by theory, it is thought that the increase in theweight of the spleen is more due to an increase in cellularity caused byan antibody immune response against this human protein rather than avascular leak. It is inferred that huKS-ala-IL2(N88R) produces lesssevere vascular leaks than huKS-ala-IL2. Table 3 provides an example ofapproximate values for the x-fold increase in organ weight relative toorgans of a control mouse:

TABLE 3 WEIGHT INCREASE (x fold) HuKS-ala-IL2 huKS-ala-IL2(N88R) ORGAN(20 μg/mouse) (100 μg/mouse) Lung 4 1.7 Spleen 3 3 Liver 1.5 1 Kidney 11

The effect of various mouse strain backgrounds, with known alterationsin their immune system make-up, was evaluated with respect to thetoxicity of these Ig-IL2 fusion proteins. Mouse strains DBA/2, Balb/C,B6.CB17-Prkdc^(scid)/SzJ (SCID), beige, and SCID/beige were used. Thefusion proteins were administered as above at a dose of 25 μg and 50 μgper mouse for huKS-ala-IL2 and at a dose of 200 μg per mouse forhuKS-ala-IL2(N88R), and mouse survival and weight was assessed over atwo week period.

In the case of huKS-ala-IL2, most mice strains gave results similar tothose seen with Balb/C mice reported above: the dose of 50 μg led toanimal death at day 5, whereas at the lower dose the animals survivedand their weights recovered to about their initial weight but did notreach the weight gains of the mock-treated control animals.Interestingly, beige mice, deficient in functional NK cells, were betterable to tolerate the high dose of 50 μg; two animals had died by day 9,but one, while it initially lost significant weight (around 25% by day7), recovered, and by day 15 had attained the body weight ofmock-treated animals and those treated at the lower dose. DBA/2 micewere more sensitive to huKS-ala-IL2; even at the lower dose, DBA/2animals died at day 5 and day 9.

With huKS-ala-IL2(N88R), the increased susceptibility of DBA/2 mice toIg-IL2 fusion proteins was also apparent: by day 8, all animals haddied, and even at half the dose (100 μg) the animals had died by day 9.Again, the fusion protein was best tolerated in beige mice, whereas theSCID/beige mice lost significant weight (remained stable at around 80%of mock-treated control by day 10).

Example 7

Efficacy of an Ig-IL2 Fusion Protein with Modified Receptor BindingCharacteristics in Treatment of Various Tumors in a Mammal

a) Treatment of a CT26/KSA subcutaneous tumor in Balb/C mice. CT26 coloncarcinoma cells, transduced with the gene encoding human KS antigen(KSA), were used to induce a subcutaneous tumor. 2×10E⁶ viable cellswere suspended in 100 μl of PBS and injected subcutaneously into thedorsa of 6 week old Balb/C mice. When tumor size reached 100–200 mm³,groups of 8 mice were subjected to one of three treatment conditions: onfive consecutive days, intravenous injections with 15 μg of eitherhuKS-ala-IL2 or of huKS-ala-IL2(N88R) diluted into 200 μl of PBS, or PBSalone, were administered. Disease progression was evaluated by measuringtumor volume twice a week for 50 days. In the control animals, tumorvolume increased steadily, reaching approximately 3500 to 6000 mm³ insize at the time of sacrifice, which was around day 32. By contrast,tumor volumes for both experimental groups remained essentially constantup to 50 days, indicating that huKS-ala-IL2(N88R) was as effective ashuKS-ala-IL2 in preventing tumor growth.

b) Treatment of a LLC/KSA subcutaneous tumor in C57BL/6 mice. In asecond tumor model, a subcutaneous tumor was induced using Lewis LungCarcinoma cells transduced with the gene encoding the KS antigen. 1×10E6viable LLC cells expressing EpCAM were suspended in 100 μl of PBS andinjected subcutaneously into the dorsa of 6–8 week old C57BL/6 mice.When tumor size reached 100–150 mm³, groups of eight mice were treatedand evaluated as above, except that administered dose was increased to20 μg per injection. In the control animals, tumor volume increasedrapidly, exceeding 6500 mm³ in 20 days; the growth of the tumor for bothexperimental conditions was retarded to the same extent, reaching 4000mm³ over the same period, indicating again that there was no differencein efficacy between treatment with huKS-ala-IL2 and huKS-ala-IL2(N88R)at the same dose.

c) Treatment of a LLC/KSA subcutaneous tumor in B6.CB17-Prkdc^(scid)/SzJmice. The fusion proteins of the invention may also be effective oncells other than mature T cells. For example, in one experiment, thefusion proteins of the invention led to retardation of tumor growth evenin mice that lack mature T-cells. These results suggest that the fusionproteins of the invention may be useful in the treatment of tumors in,for example, immunocompromised patients.

An LLC/KSA subcutaneous tumor model was evaluated in 11 week oldB6.CB17-Prkd_(scid)/SzJ mice, which are compromised in their T-cell andB-cell mediated immune response. The same treatment protocol asdescribed above was followed. Tumors in the control animals grewrapidly, to 3500 mm³ in 15 days. Both huKS-ala-IL2 andhuKS-ala-IL2(N88R) were similarly effective in retarding tumor growth toless than half that size over the same period. Moreover, the differencesin tumor growth rates between the C57BL/6 mice, which have an intactimmune system, and the B6.CB17-Prkdc^(scid)/SzJ mice, which lack T cellsand B cells, were minimal.

Furthermore, the fact that KS-ala-IL2 led to the treatment of the tumorequally well in mice with an intact immune system and in mice lackingfunctional T cells, indicated that in this tumor model the immunologicresponse operated through a non-T cell mediated mechanism. Therefore, itis valuable to maintain in a therapeutic molecule the option tostimulate an immunologic response through a variety of effector cells.In the case of KS-ala-IL2(N88R), which was as effective as KS-ala-IL2 ineither mouse background, effector cell activities that act independentlyof T cells were apparently preserved.

d) Treatment of LLC/KSA metastases to the lungs of C57BL/6 mice. LLC/KSAcells were also used in a lung metastasis model. 10×10E6 viable cellswere suspended in 200 μl PBS and injected intravenously into 6–8 weekold C57BL/6 mice. On day 4, groups of eight mice were subjected to oneof the following treatment conditions: on five consecutive days, themice were injected intravenously with 200 μl PBS, or with 20 μg ofeither KS-ala-IL2 or KS-ala-IL2(N88R) diluted into 200 μl of PBS. Theanimals were sacrificed at about day 27, and lungs were dissected andfixed in Bouin's solution. The extent of metastasis in the lungs wasevaluated by scoring the percentage of surface area covered bymetastasis and by lung weight.

Lungs of the control group had over 96% of their surface area covered bymetastases, and approximately a five-fold increase in lung weight (0.75g) over a normal lung. By contrast, lungs of mice treated withhuKS-ala-IL2 were minimally covered with metastases (5.6%), and those ofmice treated with huKS-ala-IL2(N88R) were virtually free of metastases(0%). Lungs of animals treated with huKS-ala-IL2 and huKS-ala-IL2(N88R)were of normal weight. Thus, huKS-ala-IL2(N88R) proved as efficacious ashuKS-ala-IL2 in treating the lung metastases at a dose many fold lowerthan the threshold that would affect their survival.

Example 8

KS-IL2 Variants in Combination Therapy

The effect of administering a low toxicity KS-IL2 variant, such ashuKS-ala-IL2(N88R), in conjunction with a second immuno-modulatory agentfor the treatment of tumors was investigated, employing the subcutaneoustumor model LLC/KSA in mice as described in Example 7b.

a) huKS-ala-IL2 variants and cyclophosphamide. For the combinationtherapy, cyclophosphamide was administered intraperitoneally at a doseof 75 mg/kg on day 0, at which point the tumors averaged 90 mm³, and wasfollowed by a daily administration of the fusion protein over five days(on day 1 through day 5). huKS-ala-IL2(N88R) was administered at eithera 20 μg or a 100 μg dose. Control conditions included mock-treatedanimals and animals treated either with huKS-ala-IL2 alone at a 20 μgdose, or with huKS-ala-IL2(N88R) alone at a 20 μg or a 100 μg dose.Tumors in mock-treated animals had progressed to about 5000 mm³ by day19, whereas tumors of mice treated with huKS-ala-IL2 were around 2200mm³, and of mice treated with 20 μg or 100 μg of huKS-ala-IL2(N88R) werearound 2600 mm³ and 1700 mm³ respectively. Co-administration ofcyclophosphamide resulted in a tumor of 1700 mm³ at the 20 μg dose ofhuKS-ala-IL2(N88R) and of 1250 mm³ at the higher dose, significantlysmaller than the treatment with huKS-ala-IL2 alone.

b) huKS-ala-IL2 variants and indomethacin. For the combination therapy,indomethacin was administered orally at a dose of 35 μg/mouse/day alongwith a daily administration of the fusion protein over five days (day 1through day 5). Tumors initially averaged 90 mm³. huKS-ala-IL2(N88R) wasadministered at a 20 μg dose. Control conditions included mock-treatedanimals and animals treated either with huKS-ala-IL2 alone at a 20 μgdose, or with huKS-ala-IL2(N88R) alone at a 20 μg dose. Tumors inmock-treated animals had progressed to about 5000 mm³ by day 19, whereastumors of mice treated with huKS-ala-IL2 were around 2200 mm³, and ofmice treated with 20 μg of huKS-ala-IL2(N88R) were around 2600 mm³ and1700 mm³ respectively. Co-administration of indomethacin resulted in adecrease in tumor size to 850 mm³ at the 20 μg dose ofhuKS-ala-IL2(N88R), a significantly smaller tumor than obtained bytreatment with huKS-ala-IL2 alone.

Example 9

KS-IL2 Variants with an Improved Therapeutic Index.

KS-IL2 variants are constructed with mutations at particular positionsin the IL-2 sequence. For example, substitutions are created atpositions that are likely to interface with the α subunit of IL-2receptor. A suitable residue is, for example, F42 in the mature sequenceof huIL-2. The aromatic ring structure of this amino acid is thought tostabilize the local conformation in IL-2 (Mott et al, JMB 1995,247:979), and it is found that substitutions at this position with forinstance Y, A or K in the immunocytokine lead to a molecule withprogressively decreased IL-2 receptor affinity and bioactivity. Thesemolecules are tested in animals and it is found that an increase in thetherapeutic index in the treatment of tumors is achieved when comparedwith the unaltered form of the immunocytokine. Other substitutions thatare effective are at positions R38 and K43.

Other substitutions in the IL-2 portion of the immunocytokine are in aregion that is likely to interface with the β subunit, for example, atposition E15 or L19 of the mature hu IL-2. When these residues aremutated to, for example, A or R in the immunocytokine it is found thatthe variant immunocytokines have a decreased affinity for the β subunitof the IL-2 receptor as compared to the unaltered form of theimmunocytokine. It is generally found that the effects withsubstitutions to R are more severe than with substitutions to A, whichmay be related to the bulkiness of the side chain of R. These moleculesare tested in animals and it is found that an increase in therapeuticindex in the treatment of tumors is achieved when compared to theunaltered form of the immunocytokine. Other substitutions are introducedat positions D84 and V91 and are shown also to be effective inincreasing the therapeutic index.

A substitution in the IL-2 portion of the immunocytokine that is likelyto affect a region of the molecule that interfaces with the γ subunit ofthe IL-2 receptor is introduced at position N119 of the mature hu IL-2.A more subtle immunocytokine variant is created with a mutation to A anda more disruptive mutation is created with a mutation to R. The effectof these variants is tested in animals bearing tumors and it is foundthat these variant immunocytokines do have an improved therapeutic indexas compared to the unaltered form of the immunocytokine.

It is also found that an increase in therapeutic index can be achievedby generating multiple mutations in the IL-2 immunocytokine,particularly for molecules where single mutations in the immunocytokinehave shown only a marginal or negligible increase in therapeutic index.For example, an immunocytokine containing the combination F42A withL19A, or L19A with N119A, is found to be more effective than eitherimmunocytokine variant alone. For an application involving multiplemutations, it is particularly useful to use mutations that decrease thesize of an amino acid side chain. Another substitution introduced intothe IL-2 portion of the immunocytokine is at T51 of the mature huIL-2.Whereas a mutation to A does not show an improvement in therapeuticindex, the mutation to P creates an immunocytokine with improvedtherapeutic index when compared to the unaltered form of theimmunocytokine in the treatment of tumors.

Example 10

Ig-IL2 Fusion Protein Variant huKS-ala-IL2(D20T) and Derivatives Thereof

Variants based on Ig-IL2(D20T), which contains the substitution of anaspartate to a threonine at position 20 of the mature huIL-2, weregenerated. These variants contain additional substitutions in the Igdomain, such as in the Fc portion or in the antibody targeting domains.To generate the DNA constructs encoding these molecules, procedures werefollowed essentially as described in Example 1, using a PCR approachwith construct-specific primers to introduce the mutation andappropriate cloning strategies, familiar to those reasonably skilled inthe art.

a) huKS-ala-IL2(D20T). To introduce the mutation D20T, a PCR mutagenesisapproach was used with the primer set5′-CAGCTGCAACTGGAGCATCTCCTGCTGACCCTCCAGATGATTCTGAAT-3′ (the boldnucleotides indicating the substituted codon) (SEQ ID NO: 28) and primerT3 (5′-ATTAACCCTCACTAAAGGGA-3′) (SEQ ID NO: 29), the DNA fragment wasamplified from wild-type huIL-2 DNA on a pBS plasmid and inserted into aTA vector (Invitrogen) to generate TA-IL2(D20T). Mutagenesis wasverified by sequencing. To substitute for the original IL-2 sequence inhuKS-ala-IL2, a 385 bp PvuII/XhoI fragment from TA-IL2(D20T) was clonedinto the parental immunocytokine plasmid in a triple ligation reaction.The fusion protein was expressed and purified essentially as describedin Example 3. Amino acid sequences corresponding to hu-KS heavy andlight chain variable regions are shown in SEQ ID NOs: 30 and 31respectively.

Further variants of huKS-ala-IL2(D20T) were generated, incorporating thesame PCR-derived fragment into different plasmid back-bones.

b) dI-KS-ala-IL2(D20T). A version of KS-ala-IL2 with an alterationremoving a potential T-cell epitope has been previously described. Thefusion protein was expressed and purified essentially as described inExample 3. The amino acid sequence corresponding to the heavy chain ofthe dI-KS antibody fused to the IL2(D20T) variant is depicted in SEQ IDNO: 32. SEQ ID NO: 33 and 34 correspond to the dI-KS heavy chain andlight chain variable regions respectively.

c) De-glycosylated dI-KS-ala-IL2(D20T). Enzymatic deglycosylation usingN-Glycanase was performed on the protein dI-KS-ala-IL2(D20T) essentiallyas described in Example 2.

d) dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T). The Ig-moiety for this IL-2(D20T)fusion protein was derived from the constant region of an IgG γ4subclass (SEQ ID NO: 7), which in addition retained features of the IgGγ1 hinge (SEQ ID NO: 10). Furthermore, mutations that remove potentialT-cell epitopes were introduced. Additionally, this fusion proteincontains the substitution from asparagine to glutamine, which eliminatesthe N-glycosylation site in Fc (see Example 4). The concomitantsubstitution of a phenylalanine to alanine removes the potential T-cellepitope. The fusion protein was expressed and purified essentially asdescribed in Example 3.

e) dI-NHS76(γ2h)-ala-IL2(D20T). The Ig-moiety for this IL-2(D20T) fusionprotein was derived from the constant region of an IgG γ2 subclass,which in addition retained features of the IgG γ1 hinge. In NHS76, theIg variable regions are directed against epitopes contained inDNA-histone complexes and specifically recognize necrotic centers oftumors (Williams et al, PCT WO 00/01822). Also, a mutation thateliminates a potential T-cell epitope in the variable region of thelight chain was introduced. This residue, leucine 104, lies at the CDR3V-J junction, and was substituted by a valine. The fusion protein wasexpressed and purified essentially as described in Example 3.

f) dI-NHS76(γ2h)(FN>AQ)-ala-IL2(D20T). This protein, based on theprotein of Example 10e, additionally contains the mutations thateliminate N-linked glycosylation in Fc and a potential T-cell epitope,as described in Example 10d. The fusion protein was expressed andpurified essentially as described in Example 3. In one embodiment,fusion proteins of the invention include the heavy chain sequence of theNHS76(γ2h)(FN>AQ) molecule fused to the IL2(D20T) variant, as depictedin SEQ ID NO: 35, and the light chain variable and constant regionsequence corresponding to SEQ ID NO: 36. However, the heavy chain regionof SEQ ID NO: 35 can be used in combination with any IgG light chainvariable or constant region.

g) dI-NHS76(γ4h)-ala-IL2(D20T). This protein is similar to the onedescribed in Example 10e, but contains a heavy chain derived from the γ4rather than the γ2 IgG subclass. The fusion protein was expressed andpurified essentially as described in Example 3.

h) dI-NHS76(γ4h)(FN>AQ)-ala-IL2(D20T). This protein, based on theprotein of Example 10g, additionally contains the mutations thateliminate N-linked glycosylation in Fc and a potential T-cell epitope,as described in Example 10d. The fusion protein was expressed andpurified essentially as described in Example 3. In one embodiment,fusion proteins of the invention include the heavy chain sequence of thedI-NHS76(γ4h)(FN>AQ) molecule fused to the IL-2(D20T) variant, depictedin SEQ ID NO: 37, and the light chain variable and constant regionsequence corresponding to SEQ ID NO: 36. However, the heavy chain regionof SEQ ID NO: 37 can be used in combination with any IgG light chainvariable or constant region.

The Ig moiety of a fusion protein of the invention can include domainsof heavy chain constant regions derived from any subclass of IgG,including combinations containing domains of IgG molecules derived fromdifferent species. Accordingly, the fusion proteins of the invention mayinclude hinge regions derived from any subclass of IgG, for example, ahinge region derived from IgG gamma 1 (SEQ ID NO: 10), gamma 2 (SEQ ID11) or gamma 4 (SEQ ID NO: 12).

Activity of Ig-IL2(D20T) variants in bioassays: The Ig-IL2(D20T) fusionproteins were tested in bioassays that measure the ability of cellsdependent on IL-2 for growth to proliferate, which was expressed as anED50 value (see Example 4). The assays were performed on murine CTLL-2cells or human Kit-225 cells (which express IL-2Rαβγ), and human TF-1βcells or isolated murine NK cells (which express IL-2Rβγ).

For example, in a representative experiment it was found that, comparedto huKS-ala-IL2, the ED50 value for dI-KS-ala-IL2(D20T) in IL-2Rαβγbearing cells CTLL-2 was unchanged, whereas in IL-2Rβγ bearing cellsTF-1β it was approximately 900-fold higher. The ED50 ratio, as definedin Example 4, therefore was around 150, revealing a shift ofapproximately 750-fold in selectivity towards IL-2Rαβγ bearing CTLL-2cells as compared to huKS-ala-IL2. Compared to the shift in selectivityof approximately 20,000-fold (relative to KS-ala-IL2) seen withhuKS-ala-IL2(N88R) in this pair of cell lines, the selectivity wasreduced about 10 to 20-fold for dI-KS-ala-IL2(D20T), which reflected themeasurable proliferative response obtained from IL-2Rβγ expressingcells. This trend was also apparent when human Kit 225 cells were used.As was found with other Ig-fusion proteins containing the KS antibody,deglycosylation of the antibody portion had a small but consistenteffect on reducing the activity of the fusion protein in IL-2Rαβexpressing cells.

IL-2 dependent cell proliferation was also measured in Ig-IL(D20T)variants containing a different antibody moiety. It was found that,compared to dI-NHS76(γ2)-ala-IL2, the ED50 value fordI-NHS76(γ2)-ala-IL2(D20T) in IL-2Rαβγ bearing cells Kit-225 wasincreased 3-fold, whereas in IL-2Rβγbearing cells TF-1β it was increasedapproximately 230-fold. The resultant ED50 ratio of 350 was in the samerange as was seen with dI-KS(γ4)(FN>AQ)-ala-IL2(D20T) and at least 10fold less selective than huKS-ala-IL2(N88R). Representative results areshown in Table 4.

TABLE 4 ED50 Ratio ED50 Ratio Protein TF-1β/CTLL-2 TF-1β/Kit-225dI-KS-ala-IL2(D20T) 150 3000  dI-KS(γ4) (FN > AQ)-ala-IL2(D20T) 5600*dI-NHS76(γ2)-ala-IL2(D20T) 350 *= average of different lots

Pharmacokinetics of Ig-IL2(D20T) variants: To assess the interaction ofIg-IL2 variants with cell surface Fc receptors, binding of the Ig-IL2fusion proteins to FcγR receptors was assayed in a cell-based ELISA,using U937 cells. Fusion proteins (huKS-ala-IL2, dI-huKS-ala-IL2,dI-KS-ala-IL2(D20T), and dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T)) were diluted2-fold over a range from 100 μg/ml to 780 ng/ml, incubated with thecells and binding was detected using FITC-conjugated antihuman IgG Fc AbF(ab′)₂ (Jackson ImmunoResearch, West Grove, Pa.). The concentration ofhalf-maximal binding of huKS-ala-IL2 and dI-KS-ala-IL2 for these cellswas around 5 μg/ml, and interestingly, was increased two-fold withdI-KS-ala-IL2(D20T) protein. While the introduction of the mutation thatprevents glycosylation of the Ig moiety(dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T)) reduced the binding of this protein toU973 cells 5- to 10-fold, binding was not completely abrogated.

The pharmacokinetic properties of the Ig-IL2(D20T) variants in mice wereinvestigated, essentially as described in Example 5. Surprisingly, whencompared to dI-KS-ala-IL2, the half-life of dI-KS-ala-IL2(D20T) wasdrastically reduced. Analysis of the PK profile indicated that theeffect was particularly dramatic during the α-phase: whereas 50% ofdI-KS-ala-IL2 was still available after 1 hour, only approximately 5% ofdI-KS-ala-IL2(D20T) was still present. The slopes of the β-phase of thePK profile for these proteins were similar. An essentially identical PKprofile to the one seen with dI-KS-ala-IL2(D20T) was obtained with thefusion protein dI-NHS76(γ2h)-ala-IL2(D20T), which contains an IgG ofsubclass γ2, that normally exhibits the least FcR binding affinity.Thus, the effect of the IL(D20T) protein moiety on the fusion proteinwas not limited to the antibody dI-KS.

Deglycosylation of an Ig fusion protein generally was observed to havethe effect of enhancing the α-phase of a PK profile. The effect ofenzymatic deglycosylation of dI-KS-ala-IL2(D20T) on the PK profile wastherefore investigated. In fact, the α-phase of the PK profile wasessentially restored to what had been observed with dI-KS-ala-IL2. Thesame effect was achieved when the glycosylation was abrogated bymutagenesis, as in the fusion protein dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T).It is thus likely that the effect on the PK profile is due to reducedFcR binding.

Toxicity of Ig-IL2(D20T) variants: The toxicity of Ig-IL2(D20T) variantKS(γ4h)(FN>AQ)-ala-IL2(D20T) was compared to that of dI-KS-ala-IL2 inBalb/C mice, as described in Example 6.

Both fusion proteins had a similar serum half-life in mice.dI-(γ4h)(FN>AQ)-ala-IL2(D20T) was administered in five daily doses ofeither 100 μg/mouse, 200 μg/mouse or 400 μg/mouse whereas dI-KS-ala-IL2was administered in five daily doses of 40 μg/mouse. It was found thatthe mice survived even a dose of 400 μg/mouse ofdI-KS(γ4h)(FN>AQ)-ala-IL2(D20T), whereas control mice, which receivedone tenth the dose of dI-KS-ala-IL2, had died by day 6. The body weightsof the mice treated with dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T) was slightlyaffected, dropping transiently to 97% of initial weight on day 7. Adifference of more than 10-fold in the tolerated dose may indicate asubstantial improvement in the therapeutic index.

Efficacy of Ig-IL(D20T) variants for the treatment of tumors: Theefficacy of Ig-IL2(D20T) variants was evaluated in Balb/C mice bearing asubcutaneous tumor derived from CT26/KSA cells, as described in Example7a.

The fusion protein dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T) was administered atdoses of 15 μg/mouse and 30 μg/mouse. Tumors started at an average sizeof 126 mm³ and reached sizes between 1800 mm³ and 5000 mm³ by day 28.Tumors in mice treated with 15 μg/mouse of dI-KS-ala-IL2 had grown to anaverage size of 355 mm³, while tumors in mice treated with 15 μg/mouseof dI-KS-ala-IL2(D20T) had reached an average size of 2250 mm³. This wasmost likely due to the poor PK of the molecule. Tumors in mice treatedwith dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T) at the lower dose of 15 μg/mousehad grown to some extent, to an average size of 1450 mm³; however,whereas at the 30 μg/mouse dose tumors reached an average size of 950mm³, significantly, in over half the mice the tumors had not grownappreciably. Thus, at increased doses dI-KS(γ4h)(FN>AQ)-ala-IL2(D20T)had a significant effect on inhibiting tumor growth. In fact, the doseused in this experiment was at least 12-fold lower than a maximaltolerated dose for this molecule and therefore it is likely to have animproved therapeutic index over the huKS-ala-IL2, which by comparisonwas administered at one third to one half of maximal tolerated dose.

Example 11

Relative Affinities of Wild-type and Mutant IL-2 Fusion Proteins forDifferent IL-2 Receptors

Differential affinity of the various fusion proteins of the inventionfor an IL-2Rβγ receptor relative to an IL-2Rαβγ receptor can be measuredby an assay such as a radioimmunoassay. Equal numbers of IL-2Rαβγreceptor expressing cells or IL-2Rβγ receptor expressing cells areplated on plastic plates. A dilution series is performed with an equalamount of either wild-type or mutant IL-2 fusion protein added to equalnumbers of IL-2Rαβγ receptor expressing cells or IL-2Rβγ receptorexpressing cells in order to obtain a standard curve. Unbound fusionproteins are washed away and the amount of fusion protein bound to eachcell type is detected by a radiolabelled ligand. In the case of anFc-IL-2 fusion protein, the ligand can be a molecule such as astaphylococcal protein A which binds to the Fc portion of an IgG. Theligand can also be another antibody that recognizes a portion of aparticular subclass of the IgG molecule, for example, antibodies to IgGgamma 1, IgG gamma 2 or IgG gamma 4 constant regions. Unbound ligand iswashed away and radioactivity of the plate containing either IL-2Rαβγexpresing cells bound with wild-type IL-2 fusion protein; IL-2αβγexpressing cells bound with mutant IL-2 fusion protein; IL-2Rβγexpressing cells bound with wild-type IL-2 fusion protein or IL-2Rβγexpressing cells bound with mutant fusion protein is measured on a gammacounter. The data obtained from the binding assay is normalized toaccount for the number of cells and receptors expressed on the cells.

In yet another assay, the fusion proteins themselves can be labeled,either radioactively, or non-radioactively using a variety of techniqueswell known in the art. Similar to the assay described above for alabeled ligand, either wild-type or mutant labeled fusion protein isadded to equal number of plated cells and the amount of labeled fusionprotein is measured.

The binding affinity of a fusion protein for a particular receptor ismeasured by the ratio of the concentration of the bound ligand or boundfusion protein, as described above, to the product of the concentrationof unbound ligand or unbound fusion protein and the total concentrationof the fusion protein added to each reaction. When compared to awild-type IL-2 fusion protein, certain mutations in the IL-2 moietyalter the fusion protein's relative affinity for an IL-2Rβγ receptor andan IL-2Rαβγ receptor.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

All patents, patent applications and scientific publications referencedto herein are incorporated by reference in their entirety.

1. A fusion protein comprising a non-IL-2 moiety fused to a mutant IL-2moiety wherein the mutant IL-2 moiety comprises an amino acidsubstitution changing an aspartic acid to a threonine corresponding toposition 20 of a mature human IL-2 protein consisting of the amino acidsequence set forth in SEQ ID NO:3, wherein the fusion protein exhibitsgreater selectivity than a reference protein towards cells expressing ahigh-affinity receptor, wherein said reference protein comprises thenon-IL-2 moiety fused to a non-mutant IL-2 moiety, and wherein saidselectivity is measured as a ratio of activation of cells expressingIL-2Rαβγ receptor relative to activation of cells expressing IL-2Rβγreceptor.
 2. The fusion protein of claim 1, wherein said selectivity isbetween about 0.1% to about 100% of selectivity of a protein, theprotein comprising the non-IL-2 moiety fused to a mutant human IL-2moiety, the mutant human IL-2 moiety comprising an asparagine toarginine amino acid substitution corresponding to position 88 of themature human IL-2 protein consisting of the amino acid sequence setforth in SEQ ID NO:3.
 3. The fusion protein of claim 1, wherein saidselectivity is between about 0.1% to about 30% of selectivity of aprotein, the protein comprising the non-IL-2 moiety fused to a mutanthuman IL-2 moiety, the mutant human IL-2 moiety comprising an asparagineto arginine amino acid substitution corresponding to position 88 of themature human IL-2 protein consisting of the amino acid sequence setforth in SEQ ID NO:3.
 4. The fusion protein of claim 1, wherein saidselectivity is between about 1% to about 20% of selectivity of aprotein, the protein comprising the non-IL-2 moiety fused to a mutanthuman IL-2 moiety, the mutant human IL-2 moiety comprising an asparagineto arginine amino acid substitution corresponding to position 88 of themature human IL-2 protein consisting of the amino acid sequence setforth in SEQ ID NO:3.
 5. The fusion protein of claim 1, wherein saidselectivity is between about 2% to about 10% of selectivity of aprotein, the protein comprising the non-IL-2 moiety fused to a mutanthuman IL-2 moiety, the mutant human IL-2 moiety comprising an asparagineto arginine amino acid substitution corresponding to position 88 of themature human IL-2 protein consisting of the amino acid sequence setforth in SEQ ID NO:3.
 6. The fusion protein of claim 1, wherein thecells expressing IL-2Rαβγ receptor are selected from the groupconsisting of CTLL-2, Kit 225 and mature T-cells.
 7. The fusion proteinof claim 1, wherein the cells expressing IL-2Rβγ receptor are selectedfrom the group consisting of TF-1β cells and NK cells.
 8. The fusionprotein of claim 1, wherein said fusion protein has a longer serumhalf-life than mature human IL-2 protein.
 9. The fusion protein of claim1, wherein said non-IL-2 moiety is albumin.
 10. The fusion protein ofclaim 1, wherein said non-IL-2 moiety comprises an antibody.
 11. Thefusion protein of claim 10, wherein the fusion protein comprises anamino acid sequence selected from the group consisting of the amino acidsequences set forth in SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NQ:37. 12.The fusion protein of claim 1, wherein said mutant IL-2 moiety furthercomprises an amino acid substitution changing an asparagine to argininecorresponding to position 88 of the mature human IL-2 protein consistingof the amino acid sequence set forth in SEQ ID NO:3.
 13. The fusionprotein of claim 12, wherein said mutant IL-2 moiety further comprisesan amino acid substitution changing a leucine to a threonine at position85 and an amino acid substitution changing an isoleucine to a threoninecorresponding to position 86 of the mature human IL-2 protein consistingof the amino acid sequence set forth in SEQ ID NO:3.
 14. The fusionprotein of claim 1, wherein said mutant IL-2 moiety further comprises amutation corresponding to an amino acid position of mature human IL-2protein consisting of the amino acid sequence set forth in SEQ ID NO:3,the amino acid position selected from the group consisting of lysine 8,glutamine 13, glutamic acid 15, histidine 16, leucine 19, glutamine 22,methionine 23, asparagine 26, phenylalanine 42, histidine 79, leucine80, arginine at 81, aspartic acid 84, asparagine 88, isoleucine 92 andglutamic acid
 95. 15. The fusion protein of claim 1, wherein said mutantIL-2 moiety further comprises mature human IL-2 protein with a mutationcorresponding to an amino acid position of mature human IL-2 proteinconsisting of the amino acid sequence set forth in SEQ ID NO:3, theamino acid position selected from the group consisting of leucine 25,asparagine 31, leucine 40, methionine 46, lysine 48, lysine 49,threonine 51, aspartic acid 109, glutamic acid 110, alanine 112,threonine 113, valine 115, glutamic acid 116, asparagine 119, arginine120, isoleucine 122, threonine 123, glutamine 126, serine 127, serine130 and threonine
 131. 16. The fusion protein of claim 15, wherein theamino acid mutation is an amino acid substitution changing a glutamineto an aspartic acid corresponding to position 126 of the mature humanIL-2 protein consisting of the amino acid sequence set forth in SEQ IDNO:3.
 17. The fusion protein of claim 1, wherein the non-IL-2 moietycomprises an antibody constant domain.
 18. The fusion protein of claim17, wherein said antibody constant domain comprises an IgG gamma-1constant domain, an IgG gamma-2 constant domain or an IgG gamma-4constant domain.
 19. The fusion protein of claim 17, wherein saidantibody constant domain comprises a mutation.
 20. The fusion protein ofclaim 19, wherein said antibody constant domain comprises the amino acidsequence set forth in SEQ ID NO: 1, but wherein said mutation changesthe asparagine of SEQ ID NO: 1 to a different amino acid.
 21. The fusionprotein of claim 20, wherein said asparagine is changed to a glutamine.22. The fusion protein of claim 19, wherein said antibody constantdomain comprises the amino acid sequence set forth in SEQ ID NO:2, butwherein said mutations changes the phenylalanine of SEQ ID NO:2 to analanine and changes the asparagine of SEQ ID NO:2 to glutamine.
 23. Thefusion protein of claim 10, wherein the fusion protein comprises twopeptides comprising amino acid sequences of SEQ ID NO: 35 and SEQ ID NO:36.